Amylases, nucleic acids encoding them and methods for making and using them

ABSTRACT

In one aspect, the invention is directed to polypeptides having an amylase activity, polynucleotides encoding the polypeptides, and methods for making and using these polynucleotides and polypeptides. The polypeptides of the invention can be used as amylases to catalyze the hydrolysis of starch into sugars.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/532,944, now U.S. Pat. No. 7,741,092, filed Jun. 21, 2006, which is anational phase application claiming benefit of priority under 35 U.S.C.§371 to Patent Convention Treaty (PCT) International Application SerialNo: PCT/US2003/033150, filed Oct. 15, 2003 (published as WO 2004/042006,on May 21, 2004), which claims benefit of priority to U.S. ProvisionalPatent Application Ser. No. 60/423,626, filed Oct. 31, 2002. Theaforementioned applications are explicitly incorporated herein byreference in their entirety and for all purposes.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

The entire content of the following electronic submission of thesequence listing via the USPTO EFS-WEB server, as authorized and setforth in MPEP §502.05, is incorporated herein by reference in itsentirety for all purposes. The sequence listing is identified on theelectronically filed ASCII text (.txt) file as follows:

Size File Name Date of Creation (bytes)2ndSubstSequenceListingD1530-9D1.txt Nov. 12, 2010 58.6 KB (60,093bytes)

TECHNICAL FIELD

This invention relates to molecular and cellular biology andbiochemistry. In one aspect, the invention is directed to polypeptideshaving an amylase activity, polynucleotides encoding the polypeptides,and methods for making and using these polynucleotides and polypeptides.The polypeptides of the invention can be used as amylases to catalyzethe hydrolysis of starch into sugars.

BACKGROUND

Amylase is an enzyme that catalyzes the hydrolysis of starches intosugars. Amylases can hydrolyze internal alpha-1,4-glucosidic linkages instarch, largely at random, to produce smaller molecular weightmalto-dextrins. The product of hydrolysis of one amylase, alpha-amylase(α-amylase) can be maltose, maltotriose or α-dextrin. Thesepolysaccharides can be converted to glucose by the action of otherenzymes including, for example, beta-amylase (β-amylase). Someβ-amylases hydrolyze residues at the non-reducing terminus of thepolysaccharide.

Amylases can be used commercially in the initial stages (liquefaction)of starch processing; in wet corn milling; in alcohol production; ascleaning agents in detergent matrices; in the textile industry forstarch desizing; in baking applications; in the beverage industry; inoilfields in drilling processes; in inking of recycled paper and inanimal feed. Amylases are also useful in textile desizing, brewingprocesses, starch modification in the paper and pulp industry and otherprocesses.

SUMMARY

The invention provides isolated or recombinant nucleic acids comprisinga nucleic acid sequence having at least about 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete(100%) sequence identity to SEQ ID NO:5 over a region of at least about10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050,1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650,1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2200, 2250, 2300,2350, 2400, 2450, 2500, or more, residues, wherein the nucleic acidencodes at least one polypeptide having an amylase activity, and thesequence identities are determined by analysis with a sequencecomparison algorithm or by a visual inspection.

The invention provides isolated or recombinant nucleic acids comprisinga nucleic acid sequence having at least about 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, orcomplete (100%) sequence identity to SEQ ID NO:7 over a region of atleast about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550,1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2200,2250, 2300, 2350, 2400, 2450, 2500, or more, residues, wherein thenucleic acid encodes at least one polypeptide having an amylaseactivity, and the sequence identities are determined by analysis with asequence comparison algorithm or by a visual inspection.

The invention provides isolated or recombinant nucleic acids comprisinga nucleic acid sequence having at least about 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more, or complete (100%), sequence identity to SEQ IDNO:11 over a region of at least about 10, 15, 20, 25, 30, 35, 40, 45,50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950,2000, 2050, 2100, 2200, 2250, 2300, 2350, 2400, 2450, 2500, or more, ormore, residues, wherein the nucleic acid encodes at least onepolypeptide having an amylase activity, and the sequence identities aredetermined by analysis with a sequence comparison algorithm or by avisual inspection.

The invention provides isolated or recombinant nucleic acids comprisinga nucleic acid sequence having at least about 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, orcomplete (100%) sequence identity to SEQ ID NO:13 over a region of atleast about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550,1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2200,2250, 2300, 2350, 2400, 2450, 2500, or more, residues, wherein thenucleic acid encodes at least one polypeptide having an amylaseactivity, and the sequence identities are determined by analysis with asequence comparison algorithm or by a visual inspection.

The invention provides isolated or recombinant nucleic acids comprisinga nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more, or complete (100%) sequence identity to SEQ ID NO:15 overa region of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400,1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000,2050, 2100, 2200, 2250, 2300, 2350, 2400, 2450, 2500, or more, residues,wherein the nucleic acid encodes at least one polypeptide having anamylase activity, and the sequence identities are determined by analysiswith a sequence comparison algorithm or by a visual inspection.

In one aspect, the invention provides isolated or recombinant nucleicacids, wherein the nucleic acid sequence comprises a sequence as setforth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13 or SEQ ID NO:15, or fragments orsubsequences thereof.

In one aspect, the invention provides isolated or recombinant nucleicacids, wherein the nucleic acid sequence encodes a polypeptidecomprising a polypeptide having a sequence as set forth in SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:14 or SEQ ID NO:16, or a subsequence or fragment thereof.

In one aspect, the sequence comparison algorithm is a BLAST version2.2.2 algorithm where a filtering setting is set to blastall -p blastp-d “nr pataa” -F F, and all other options are set to default.

In one aspect, the amylase activity comprises hydrolyzing glucosidicbonds. The amylase activity can comprise a glucoamylase activity, a1,4-α-D-glucan glucohydrolase activity, an α-amylase activity, anexoamylase activity, or a β-amylase activity. In one aspect, theglucosidic bonds comprise an α-1,4-glucosidic bond. In another aspect,the glucosidic bonds comprise an α-1,6-glucosidic bond. In one aspect,the amylase activity comprises hydrolyzing glucosidic bonds in starch,e.g., liquefied starch. The amylase activity can further comprisehydrolyzing glucosidic bonds into maltodextrines. In one aspect, theamylase activity comprises cleaving a maltose or a D-glucose unit fromnon-reducing end of the starch.

In one aspect, the isolated or recombinant nucleic acid encodes apolypeptide having an amylase activity which is thermostable. Thepolypeptide can retain an amylase activity under conditions comprising atemperature range of between about 37° C. to about 95° C.; between about55° C. to about 85° C., between about 70° C. to about 95° C., or,between about 90° C. to about 95° C.

In another aspect, the isolated or recombinant nucleic acid encodes apolypeptide having an amylase activity which is thermotolerant. Thepolypeptide can retain an amylase activity after exposure to atemperature in the range from greater than 37° C. to about 95° C. oranywhere in the range from greater than 55° C. to about 85° C. In oneaspect, the polypeptide retains an amylase activity after exposure to atemperature in the range from greater than 90° C. to about 95° C. at pH4.5.

The invention provides isolated or recombinant nucleic acids comprisinga sequence that hybridizes under stringent conditions to a nucleic acidcomprising a sequence as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13 or SEQ IDNO:15, or fragments or subsequences thereof. In one aspect, the nucleicacid encodes a polypeptide having an amylase activity. The nucleic acidcan be at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450,1500 or more residues in length or the full length of the gene ortranscript. In one aspect, the stringent conditions include a wash stepcomprising a wash in 0.2×SSC at a temperature of about 65° C. for about15 minutes.

The invention provides a nucleic acid probe for identifying a nucleicacid encoding a polypeptide having an amylase activity, wherein theprobe comprises at least about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90,100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,800, 850, 900, 950, 1000 or more, consecutive bases of a sequencecomprising a sequence as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13 or SEQ IDNO:15, or fragments or subsequences thereof, wherein the probeidentifies the nucleic acid by binding or hybridization. The probe cancomprise an oligonucleotide comprising at least about 10 to 50, about 20to 60, about 30 to 70, about 40 to 80, or about 60 to 100 consecutivebases of a sequence comprising a sequence of the invention, e.g., asequence as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13 or SEQ ID NO:15, orfragments or subsequences thereof.

The invention provides a nucleic acid probe for identifying a nucleicacid encoding a polypeptide having an amylase activity, wherein theprobe comprises/consists of a nucleic acid of the invention (whichincludes both sense and antisense strands) that is at least about 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or moreresidues. In one aspect, the probe has at least about 99.5% or moresequence identity to SEQ ID NO:1, wherein the sequence identities aredetermined by analysis with a sequence comparison algorithm or by visualinspection.

The invention provides a nucleic acid probe for identifying a nucleicacid encoding a polypeptide having an amylase activity, wherein theprobe comprises/consists of a sequence of the invention at least about10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or moreresidues. In one aspect, the probe comprises at least about 99.5% ormore sequence identity to SEQ ID NO:3, wherein the sequence identitiesare determined by analysis with a sequence comparison algorithm or byvisual inspection.

In one aspect, the invention provides a nucleic acid probe foridentifying a nucleic acid encoding a polypeptide having an amylaseactivity, wherein the probe comprises a nucleic acid sequence having atleast about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more,or complete (100%), sequence identity to SEQ ID NO:5 over a region of atleast about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100,1200, 1300, 1400, 1500 or more residues, wherein the sequence identitiesare determined by analysis with a sequence comparison algorithm or by avisual inspection.

The invention provides a nucleic acid probe for identifying a nucleicacid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%)sequence identity to SEQ ID NO:7 over a region of at least about 100,200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400,1500 or more residues, wherein the sequence identities are determined byanalysis with a sequence comparison algorithm or by a visual inspection.

The invention provides a nucleic acid probe for identifying a nucleicacid encoding a polypeptide having an amylase activity, wherein theprobe comprises a nucleic acid comprising a sequence at least about 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or moreresidues having at least about 99.5% or more sequence identity to SEQ IDNO:9, wherein the sequence identities are determined by analysis with asequence comparison algorithm or by visual inspection.

The invention provides a nucleic acid probe for identifying a nucleicacid sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more, or complete sequence identity to SEQ IDNO:11 over a region of at least about 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, 1100, 1200, 1300, 1400, 1500 or more residues, whereinthe sequence identities are determined by analysis with a sequencecomparison algorithm or by a visual inspection.

The invention provides a nucleic acid probe for identifying a nucleicacid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, orcomplete sequence identity to SEQ ID NO:13 over a region of at leastabout 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200,1300, 1400, 1500 or more residues, wherein the sequence identities aredetermined by analysis with a sequence comparison algorithm or by avisual inspection.

The invention provides a nucleic acid probe for identifying a nucleicacid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more, or complete sequence identity to SEQ ID NO:15 over aregion of at least about 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 1100, 1200, 1300, 1400, 1500 or more residues, wherein thesequence identities are determined by analysis with a sequencecomparison algorithm or by visual inspection.

The probe can comprise an oligonucleotide comprising at least about 10to 50, about 20 to 60, about 30 to 70, about 40 to 80, or about 60 to100 consecutive bases of a nucleic acid sequence as set forth in SEQ IDNO:1, or a subsequence thereof, a sequence as set forth in SEQ ID NO:5,or a subsequence thereof, a sequence as set forth in SEQ ID NO:7, or asubsequence thereof, a sequence as set forth in SEQ ID NO:11, or asubsequence thereof, a sequence as set forth in SEQ ID NO:13, or asubsequence thereof, a sequence as set forth in SEQ ID NO:15, or asubsequence thereof.

The invention provides an amplification primer sequence pair foramplifying a nucleic acid encoding a polypeptide having an amylaseactivity, wherein the primer pair is capable of amplifying a nucleicacid comprising a sequence as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13 or SEQ IDNO:15, or fragments or subsequences thereof. One or each member of theamplification primer sequence pair can comprise an oligonucleotidecomprising at least about 10 to 50 consecutive bases of the sequence, orabout 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30 or more consecutive bases of the sequence.

The invention provides amplification primer pairs, wherein the primerpair comprises a first member having a sequence as set forth by aboutthe first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30 or more residues of a nucleic acid of theinvention, and a second member having a sequence as set forth by aboutthe first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30 or more residues of the complementary strand ofthe first member.

The invention provides amylase-encoding nucleic acids generated byamplification, e.g., polymerase chain reaction (PCR), using anamplification primer pair of the invention. The invention providesamylases generated by amplification, e.g., polymerase chain reaction(PCR), using an amplification primer pair of the invention. Theinvention provides methods of making amylases by amplification, e.g.,polymerase chain reaction (PCR), using an amplification primer pair ofthe invention. In one aspect, the amplification primer pair amplifies anucleic acid from a library, e.g., a gene library, such as anenvironmental library.

The invention provides methods of amplifying a nucleic acid encoding apolypeptide having an amylase activity comprising amplification of atemplate nucleic acid with an amplification primer sequence pair capableof amplifying a nucleic acid sequence as set forth in SEQ ID NO:1, SEQID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13 or SEQ ID NO:15, or fragments or subsequences thereof.

The invention provides expression cassettes comprising a nucleic acid ofthe invention, e.g., a nucleic acid comprising: (i) a sequence as setforth in SEQ ID NO:1, a nucleic acid sequence having at least 90%sequence identity to SEQ ID NO:5 over a region of at least about 100residues, a nucleic acid sequence having at least 60% sequence identityto SEQ ID NO:7 over a region of at least about 100 residues, a nucleicacid sequence having at least 50% sequence identity to SEQ ID NO:11 overa region of at least about 100 residues, a nucleic acid sequence havingat least 70% sequence identity to SEQ ID NO:13 over a region of at leastabout 100 residues, or a nucleic acid sequence having at least 80%sequence identity to SEQ ID NO:15 over a region of at least about 100residues, wherein the sequence identities are determined by analysiswith a sequence comparison algorithm or by a visual inspection; or, (ii)a nucleic acid that hybridizes under stringent conditions to a nucleicacid comprising a sequence as set forth in SEQ ID NO:1, or a subsequencethereof, a sequence as set forth in SEQ ID NO:5, or a subsequencethereof, a sequence as set forth in SEQ ID NO:7, or a subsequencethereof, a sequence as set forth in SEQ ID NO:11, or a subsequencethereof, a sequence as set forth in SEQ ID NO:13, or a subsequencethereof, a sequence as set forth in SEQ ID NO:15, or a subsequencethereof.

In one aspect, the expression cassette can comprise the nucleic acidthat is operably linked to a promoter. The promoter can be a viral,bacterial, mammalian or plant promoter. In one aspect, the plantpromoter can be a potato, rice, corn, wheat, tobacco or barley promoter.The promoter can be a constitutive promoter. The constitutive promotercan comprise CaMV35S. In another aspect, the promoter can be aninducible promoter. In one aspect, the promoter can be a tissue-specificpromoter or a environmentally regulated or a developmentally regulatedpromoter. Thus, the promoter can be, e.g., a seed-specific, aleaf-specific, a root-specific, a stem-specific or an abscission-inducedpromoter. In one aspect, the expression cassette can further comprise aplant or plant virus expression vector.

The invention provides vectors comprising a nucleic acid of theinvention, e.g., a nucleic acid comprising (i) a sequence as set forthin SEQ ID NO:1, a nucleic acid sequence having at least 90% sequenceidentity to SEQ ID NO:5 over a region of at least about 100 residues, anucleic acid sequence having at least 60% sequence identity to SEQ IDNO:7 over a region of at least about 100 residues, a nucleic acidsequence having at least 50% sequence identity to SEQ ID NO:11 over aregion of at least about 100 residues, a nucleic acid sequence having atleast 70% sequence identity to SEQ ID NO:13 over a region of at leastabout 100 residues, or a nucleic acid sequence having at least 80%sequence identity to SEQ ID NO:15 over a region of at least about 100residues, wherein the sequence identities are determined by analysiswith a sequence comparison algorithm or by a visual inspection; or, (ii)a nucleic acid that hybridizes under stringent conditions to a nucleicacid comprising a sequence as set forth in SEQ ID NO:1, or a subsequencethereof, a sequence as set forth in SEQ ID NO:5, or a subsequencethereof, a sequence as set forth in SEQ ID NO:7, or a subsequencethereof, a sequence as set forth in SEQ ID NO:11, or a subsequencethereof, a sequence as set forth in SEQ ID NO:13, or a subsequencethereof, a sequence as set forth in SEQ ID NO:15, or a subsequencethereof.

The invention provides cloning vehicles comprising an expressioncassette (e.g., a vector) of the invention or a nucleic acid of theinvention. The cloning vehicle can be a viral vector, a plasmid, aphage, a phagemid, a cosmid, a fosmid, a bacteriophage or an artificialchromosome. The viral vector can comprise an adenovirus vector, aretroviral vector or an adeno-associated viral vector. The cloningvehicle can comprise a bacterial artificial chromosome (BAC), a plasmid,a bacteriophage P1-derived vector (PAC), a yeast artificial chromosome(YAC), or a mammalian artificial chromosome (MAC).

The invention provides transformed cell comprising a nucleic acid of theinvention or an expression cassette (e.g., a vector) of the invention,or a cloning vehicle of the invention, e.g., a nucleic acid comprising(i) a sequence as set forth in SEQ ID NO:1, a nucleic acid sequencehaving at least 90% sequence identity to SEQ ID NO:5 over a region of atleast about 100 residues, a nucleic acid sequence having at least 60%sequence identity to SEQ ID NO:7 over a region of at least about 100residues, a nucleic acid sequence having at least 50% sequence identityto SEQ ID NO:11 over a region of at least about 100 residues, a nucleicacid sequence having at least 70% sequence identity to SEQ ID NO:13 overa region of at least about 100 residues, or a nucleic acid sequencehaving at least 80% sequence identity to SEQ ID NO:15 over a region ofat least about 100 residues, wherein the sequence identities aredetermined by analysis with a sequence comparison algorithm or by avisual inspection; or, (ii) a nucleic acid that hybridizes understringent conditions to a nucleic acid comprising a sequence as setforth in SEQ ID NO:1, or a subsequence thereof, a sequence as set forthin SEQ ID NO:5, or a subsequence thereof, a sequence as set forth in SEQID NO:7, or a subsequence thereof, a sequence as set forth in SEQ IDNO:11, or a subsequence thereof, a sequence as set forth in SEQ IDNO:13, or a subsequence thereof, a sequence as set forth in SEQ IDNO:15, or a subsequence thereof. In one aspect, the transformed cell canbe a bacterial cell, a mammalian cell, a fungal cell, a yeast cell, aninsect cell or a plant cell. In one aspect, the plant cell can be apotato, wheat, rice, corn, tobacco or barley cell.

The invention provides transgenic non-human animals comprising a nucleicacid of the invention or an expression cassette (e.g., a vector) of theinvention. In one aspect, the animal is a mouse.

The invention provides transgenic plants comprising a nucleic acid ofthe invention or an expression cassette (e.g., a vector) of theinvention. The transgenic plant can be a corn plant, a potato plant, atomato plant, a wheat plant, an oilseed plant, a rapeseed plant, asoybean plant, a rice plant, a barley plant or a tobacco plant.

The invention provides transgenic seeds comprising a nucleic acid of theinvention or an expression cassette (e.g., a vector) of the invention.The transgenic seed can be a corn seed, a wheat kernel, an oilseed, arapeseed, a soybean seed, a palm kernel, a sunflower seed, a sesameseed, a peanut or a tobacco plant seed.

The invention provides an antisense oligonucleotide comprising a nucleicacid sequence complementary to or capable of hybridizing under stringentconditions to a nucleic acid of the invention, e.g., (i) a sequence asset forth in SEQ ID NO:1, or a subsequence thereof; a sequence as setforth in SEQ ID NO:3, or a subsequence thereof; a nucleic acid sequencehaving at least 90% sequence identity to SEQ ID NO:5 over a region of atleast about 100 residues, or a subsequence thereof; a nucleic acidsequence having at least 60% sequence identity to SEQ ID NO:7 over aregion of at least about 100 residues, or a subsequence thereof; anucleic acid sequence having at least 50% sequence identity to SEQ IDNO:11 over a region of at least about 100 residues, or a subsequencethereof; a nucleic acid sequence having at least 70% sequence identityto SEQ ID NO:13 over a region of at least about 100 residues, or asubsequence thereof; or a nucleic acid sequence having at least 80%sequence identity to SEQ ID NO:15 over a region of at least about 100residues, or a subsequence thereof; wherein the sequence identities aredetermined by analysis with a sequence comparison algorithm or by avisual inspection; or, (ii) a nucleic acid that hybridizes understringent conditions to a nucleic acid comprising a sequence as setforth in SEQ ID NO:1, or a subsequence thereof, a sequence as set forthin SEQ ID NO:5, or a subsequence thereof, a sequence as set forth in SEQID NO:7, or a subsequence thereof, a sequence as set forth in SEQ IDNO:11, or a subsequence thereof, a sequence as set forth in SEQ IDNO:13, or a subsequence thereof, a sequence as set forth in SEQ IDNO:15, or a subsequence thereof. The antisense oligonucleotide can bebetween about 10 to 50, about 20 to 60, about 30 to 70, about 40 to 80,or about 60 to 100 bases in length.

The invention provides methods of inhibiting the translation of anamylase message in a cell comprising administering to the cell orexpressing in the cell an antisense oligonucleotide comprising a nucleicacid sequence complementary to or capable of hybridizing under stringentconditions to a nucleic acid of the invention.

The invention provides methods of inhibiting the translation of anamylase message in a cell comprising administering to the cell orexpressing in the cell an antisense oligonucleotide comprising a nucleicacid sequence complementary to or capable of hybridizing under stringentconditions to a nucleic acid of the invention. The invention providesdouble-stranded inhibitory RNA (RNAi) molecules comprising a subsequenceof a sequence of the invention. In one aspect, the RNAi is about 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in length.The invention provides methods of inhibiting the expression of anamylase in a cell comprising administering to the cell or expressing inthe cell a double-stranded inhibitory RNA (iRNA), wherein the RNAcomprises a subsequence of a sequence of the invention.

The invention provides an isolated or recombinant polypeptidecomprising: (a) an amino acid sequence having at least 99.5% or more,identity to SEQ ID NO:2 over a region of at least about 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, ormore, residues, or, an amino acid sequence as set forth in SEQ ID NO:2,an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more, or complete (100%) identity to SEQ ID NO:6over a region of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, or more, residues; an aminoacid sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more, or complete (100%) identity to SEQ ID NO:8over a region of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, or more, residues, an aminoacid sequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore, or complete (100%) identity to SEQ ID NO:12 over a region of atleast about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, or more, residues; an amino acid sequencehaving at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more, or complete (100%) identity to SEQ IDNO:14 over a region of at least about 10, 20, 30, 40, 50, 60, 70, 80,90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or more, residues;or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore, or complete (100%) identity to SEQ ID NO:16 over a region of atleast about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, or more, residues, or (b) a polypeptide encodedby a nucleic acid comprising (i) a nucleic acid sequence as set forth inSEQ ID NO:1, a nucleic acid sequence having at least 90% sequenceidentity to SEQ ID NO:5 over a region of at least about 100 residues, anucleic acid sequence having at least 60% sequence identity to SEQ IDNO:7 over a region of at least about 100 residues, a nucleic acidsequence having at least 50% sequence identity to SEQ ID NO:11 over aregion of at least about 100 residues, a nucleic acid sequence having atleast 70% sequence identity to SEQ ID NO:13 over a region of at leastabout 100 residues, or a nucleic acid sequence having at least 80%sequence identity to SEQ ID NO:15 over a region of at least about 100residues, wherein the sequence identities are determined by analysiswith a sequence comparison algorithm or by a visual inspection; or, (ii)a nucleic acid that hybridizes under stringent conditions to a nucleicacid comprising a sequence as set forth in SEQ ID NO:1, or a subsequencethereof, a sequence as set forth in SEQ ID NO:5, or a subsequencethereof, a sequence as set forth in SEQ ID NO:7, or a subsequencethereof, a sequence as set forth in SEQ ID NO:11, or a subsequencethereof, a sequence as set forth in SEQ ID NO:13, or a subsequencethereof, a sequence as set forth in SEQ ID NO:15, or a subsequencethereof. In one aspect, the polypeptide has an amylase activity.

The invention provides an isolated or recombinant polypeptide orpeptide, wherein the polypeptide or peptide comprises an amino acidsequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 or SEQ ID NO:16, or asubsequence or fragment thereof. Another aspect of the inventionprovides an isolated or recombinant polypeptide or peptide including atleast 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95 or 100 or more consecutive bases of a polypeptide or peptidesequence of the invention, sequences substantially identical thereto,and the sequences complementary thereto. The peptide can be, e.g., animmunogenic fragment, a motif (e.g., a binding site), a signal sequence(e.g., as in Table 4), a prepro sequence or an active site.

In one aspect, the amylase activity comprises hydrolyzing glucosidicbonds. The amylase activity can comprise a glucoamylase activity, a1,4-α-D-glucan glucohydrolase activity, an α-amylase activity, anexoamylase activity, or a β-amylase activity. In one aspect, theglucosidic bonds comprise an α-1,4-glucosidic bond or anα-1,6-glucosidic bond. In one aspect, the amylase activity compriseshydrolyzing glucosidic bonds in starch, e.g., liquefied starch. Theamylase activity can further comprise hydrolyzing glucosidic bonds intomaltodextrines. In one aspect, the amylase activity comprises cleaving amaltose or a D-glucose unit from non-reducing end of the starch.

In one aspect, the amylase activity can be thermostable. The polypeptidecan retain an amylase activity under conditions comprising a temperaturerange of between about 37° C. to about 95° C., between about 55° C. toabout 85° C., between about 70° C. to about 95° C., or between about 90°C. to about 95° C. In another aspect, the amylase activity can bethermotolerant. The polypeptide can retain an amylase activity afterexposure to a temperature in the range from greater than 37° C. to about95° C., or in the range from greater than 55° C. to about 85° C. In oneaspect, the polypeptide can retain an amylase activity after exposure toa temperature in the range from greater than 90° C. to about 95° C. atpH 4.5.

In one aspect, the isolated or recombinant polypeptide can comprise thepolypeptide of the invention that lacks a signal sequence. In oneaspect, the isolated or recombinant polypeptide can comprise thepolypeptide of the invention comprising a heterologous signal sequence,such as a heterologous amylase or non-amylase signal sequence. In oneaspect, the invention provides a signal sequence comprising a peptidecomprising/consisting of a sequence as set forth in residues 1 to 12, 1to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1to 28, 1 to 30, 1 to 31, 1 to 32, 1 to 33, 1 to 34, 1 to 35, 1 to 36, 1to 37, 1 to 38, 1 to 39, 1 to 40, 1 to 41, 1 to 42, 1 to 43, 1 to 44 (ora longer peptide) of a polypeptide of the invention.

In one aspect, the invention provides chimeric proteins comprising afirst domain comprising a signal sequence of the invention and at leasta second domain. The protein can be a fusion protein. The second domaincan comprise an enzyme. The enzyme can be an amylase or another enzyme.

The invention provides chimeric polypeptides comprising at least a firstdomain comprising signal peptide (SP), a prepro sequence and/or acatalytic domain (CD) of the invention and at least a second domaincomprising a heterologous polypeptide or peptide, wherein theheterologous polypeptide or peptide is not naturally associated with thesignal peptide (SP), prepro sequence and/or catalytic domain (CD). Inone aspect, the heterologous polypeptide or peptide is not an amylase.The heterologous polypeptide or peptide can be amino terminal to,carboxy terminal to or on both ends of the signal peptide (SP), preprosequence and/or catalytic domain (CD).

The invention provides isolated or recombinant nucleic acids encoding achimeric polypeptide, wherein the chimeric polypeptide comprises atleast a first domain comprising signal peptide (SP), a prepro domainand/or a catalytic domain (CD) of the invention and at least a seconddomain comprising a heterologous polypeptide or peptide, wherein theheterologous polypeptide or peptide is not naturally associated with thesignal peptide (SP), prepro domain and/or catalytic domain (CD).

In one aspect, the amylase activity comprises a specific activity atabout 37° C. in the range from about 100 to about 1000 units permilligram of protein. In another aspect, the amylase activity comprisesa specific activity from about 500 to about 750 units per milligram ofprotein. Alternatively, the amylase activity comprises a specificactivity at 37° C. in the range from about 500 to about 1200 units permilligram of protein. In one aspect, the amylase activity comprises aspecific activity at 37° C. in the range from about 750 to about 1000units per milligram of protein. In another aspect, the thermotolerancecomprises retention of at least half of the specific activity of theamylase at 37° C. after being heated to the elevated temperature.Alternatively, the thermotolerance can comprise retention of specificactivity at 37° C. in the range from about 500 to about 1200 units permilligram of protein after being heated to the elevated temperature.

The invention provides the isolated or recombinant polypeptide of theinvention, wherein the polypeptide comprises at least one glycosylationsite. In one aspect, glycosylation can be an N-linked glycosylation. Inone aspect, the polypeptide can be glycosylated after being expressed ina P. pastoris or a S. pombe.

In one aspect, the polypeptide can retain an amylase activity underconditions comprising about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5 or pH 4or less. In another aspect, the polypeptide can retain an amylaseactivity under conditions comprising about pH 7, pH 7.5 pH 8.0, pH 8.5,pH 9, pH 9.5, pH 10, pH 10.5 or pH 11 or more. In one aspect, thepolypeptide can retain an amylase activity after exposure to conditionscomprising about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5 or pH 4 or less. Inanother aspect, the polypeptide can retain an amylase activity afterexposure to conditions comprising about pH 7, pH 7.5 pH 8.0, pH 8.5, pH9, pH 9.5, pH 10, pH 10.5 or pH 11 or more.

The invention provides protein preparations comprising a polypeptide ofthe invention, wherein the protein preparation comprises a liquid, asolid or a gel.

The invention provides heterodimers comprising a polypeptide of theinvention and a second protein or domain. The second member of theheterodimer can be a different amylase, a different enzyme or anotherprotein. In one aspect, the second domain can be a polypeptide and theheterodimer can be a fusion protein. In one aspect, the second domaincan be an epitope or a tag. In one aspect, the invention provideshomodimers comprising a polypeptide of the invention.

The invention provides immobilized polypeptides having an amylaseactivity, wherein the polypeptide comprises a polypeptide of theinvention, a polypeptide encoded by a nucleic acid of the invention, ora polypeptide comprising a polypeptide of the invention and a seconddomain. In one aspect, the polypeptide can be immobilized on a cell, ametal, a resin, a polymer, a ceramic, a glass, a microelectrode, agraphitic particle, a bead, a gel, a plate, an array or a capillarytube.

The invention provides arrays comprising an immobilized nucleic acid ofthe invention. The invention provides arrays comprising an antibody ofthe invention.

The invention provides isolated or recombinant antibodies thatspecifically bind to a polypeptide of the invention or to a polypeptideencoded by a nucleic acid of the invention. The antibody can be amonoclonal or a polyclonal antibody. The invention provides hybridomascomprising an antibody of the invention, e.g., an antibody thatspecifically binds to a polypeptide of the invention or to a polypeptideencoded by a nucleic acid of the invention.

The invention provides food supplements for an animal comprising apolypeptide of the invention or a polypeptide encoded by the nucleicacid of the invention, e.g., a polypeptide comprising an amino acidsequence having at least 90% identity to SEQ ID NO:2, SEQ ID NO:4, SEQID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 or SEQ IDNO:16, or a subsequence or fragment thereof over a region of at leastabout 100 residues; or a polypeptide encoded by a nucleic acidcomprising a nucleic acid sequence having at least 90% identity to SEQID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13 or SEQ ID NO:15 or a nucleic acid that hybridizesunder stringent conditions to a nucleic acid comprising a sequence asset forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13 or SEQ ID NO:15, or a subsequencethereof. In one aspect, the polypeptide can be glycosylated.

The invention provides edible enzyme delivery matrices comprising apolypeptide of the invention or a polypeptide encoded by the nucleicacid of the invention, e.g., a polypeptide comprising an amino acidsequence having at least 90% identity to SEQ ID NO:2, SEQ ID NO:4, SEQID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 or SEQ IDNO:16, or a subsequence or fragment thereof over a region of at leastabout 100 residues; or a polypeptide encoded by a nucleic acidcomprising a nucleic acid sequence having at least 90% identity to SEQID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13 or SEQ ID NO:15 or a nucleic acid that hybridizesunder stringent conditions to a nucleic acid comprising a sequence asset forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13 or SEQ ID NO:15, or a subsequencethereof. In one aspect, the delivery matrix comprises a pellet. In oneaspect, the polypeptide can be glycosylated. In one aspect, the amylaseactivity is thermotolerant. In another aspect, the amylase activity isthermostable.

The invention provides method of isolating or identifying a polypeptidehaving an amylase activity comprising the steps of: (a) providing anantibody of the invention; (b) providing a sample comprisingpolypeptides; and (c) contacting the sample of step (b) with theantibody of step (a) under conditions wherein the antibody canspecifically bind to the polypeptide, thereby isolating or identifying apolypeptide having an amylase activity.

The invention provides methods of making an anti-amylase antibodycomprising administering to a non-human animal a nucleic acid of theinvention or a polypeptide of the invention or subsequences thereof inan amount sufficient to generate a humoral immune response, therebymaking an anti-amylase antibody. The invention provides methods ofmaking an anti-amylase immune comprising administering to a non-humananimal a nucleic acid of the invention or a polypeptide of the inventionor subsequences thereof in an amount sufficient to generate an immuneresponse.

The invention provides methods of producing a recombinant polypeptidecomprising the steps of: (a) providing a nucleic acid of the inventionoperably linked to a promoter; and (b) expressing the nucleic acid ofstep (a) under conditions that allow expression of the polypeptide,thereby producing a recombinant polypeptide. In one aspect, the methodcan further comprise transforming a host cell with the nucleic acid ofstep (a) followed by expressing the nucleic acid of step (a), therebyproducing a recombinant polypeptide in a transformed cell.

The invention provides methods for identifying a polypeptide having anamylase activity comprising the following steps: (a) providing apolypeptide of the invention; or a polypeptide encoded by a nucleic acidof the invention; (b) providing an amylase substrate; and (c) contactingthe polypeptide or a fragment or variant thereof of step (a) with thesubstrate of step (b) and detecting a decrease in the amount ofsubstrate or an increase in the amount of a reaction product, wherein adecrease in the amount of the substrate or an increase in the amount ofthe reaction product detects a polypeptide having an amylase activity.In one aspect, the substrate can be a starch, e.g., a liquefied starch.

The invention provides methods for identifying an amylase substratecomprising the following steps: (a) providing a polypeptide of theinvention; or a polypeptide encoded by a nucleic acid of the invention;(b) providing a test substrate; and (c) contacting the polypeptide ofstep (a) with the test substrate of step (b) and detecting a decrease inthe amount of substrate or an increase in the amount of reactionproduct, wherein a decrease in the amount of the substrate or anincrease in the amount of a reaction product identifies the testsubstrate as an amylase substrate.

The invention provides methods of determining whether a test compoundspecifically binds to a polypeptide comprising the following steps: (a)expressing a nucleic acid or a vector comprising the nucleic acid underconditions permissive for translation of the nucleic acid to apolypeptide, wherein the nucleic acid comprises a nucleic acid of theinvention, or, providing a polypeptide of the invention; (b) providing atest compound; (c) contacting the polypeptide with the test compound;and (d) determining whether the test compound of step (b) specificallybinds to the polypeptide.

The invention provides methods for identifying a modulator of an amylaseactivity comprising the following steps: (a) providing a polypeptide ofthe invention or a polypeptide encoded by a nucleic acid of theinvention, including a nucleic acid having at least 90% or more sequenceidentity to SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:9; (b) providing atest compound; (c) contacting the polypeptide of step (a) with the testcompound of step (b) and measuring an activity of the amylase, wherein achange in the amylase activity measured in the presence of the testcompound compared to the activity in the absence of the test compoundprovides a determination that the test compound modulates the amylaseactivity. In one aspect, the amylase activity can be measured byproviding an amylase substrate and detecting a decrease in the amount ofthe substrate or an increase in the amount of a reaction product, or, anincrease in the amount of the substrate or a decrease in the amount of areaction product. A decrease in the amount of the substrate or anincrease in the amount of the reaction product with the test compound ascompared to the amount of substrate or reaction product without the testcompound identifies the test compound as an activator of amylaseactivity. An increase in the amount of the substrate or a decrease inthe amount of the reaction product with the test compound as compared tothe amount of substrate or reaction product without the test compoundidentifies the test compound as an inhibitor of amylase activity.

The invention provides computer systems comprising a processor and adata storage device wherein said data storage device has stored thereona polypeptide sequence or a nucleic acid sequence of the invention, or apolypeptide encoded by a nucleic acid of the invention. In one aspect,the computer system can further comprise a sequence comparison algorithmand a data storage device having at least one reference sequence storedthereon. In another aspect, the sequence comparison algorithm comprisesa computer program that indicates polymorphisms. In one aspect, thecomputer system can further comprise an identifier that identifies oneor more features in said sequence. The invention provides computerreadable media having stored thereon a polypeptide sequence or a nucleicacid sequence of the invention; or a polypeptide encoded by a nucleicacid of the invention. The invention provides methods for identifying afeature in a sequence comprising the steps of: (a) reading the sequenceusing a computer program which identifies one or more features in asequence, wherein the sequence comprises a polypeptide sequence or anucleic acid sequence of the invention; and (b) identifying one or morefeatures in the sequence with the computer program.

The invention provides methods for comparing a first sequence to asecond sequence comprising the steps of: (a) reading the first sequenceand the second sequence through use of a computer program which comparessequences, wherein the first sequence comprises a polypeptide sequenceor a nucleic acid sequence of the invention; and (b) determiningdifferences between the first sequence and the second sequence with thecomputer program. The step of determining differences between the firstsequence and the second sequence can further comprise the step ofidentifying polymorphisms. In one aspect, the method can furthercomprise an identifier that identifies one or more features in asequence. In another aspect, the method can comprise reading the firstsequence using a computer program and identifying one or more featuresin the sequence.

The invention provides methods for isolating or recovering a nucleicacid encoding a polypeptide having an amylase activity from anenvironmental sample comprising the steps of: (a) providing anamplification primer sequence pair for amplifying a nucleic acidencoding a polypeptide having an amylase activity, wherein the primerpair is capable of amplifying a nucleic acid of the invention, e.g., SEQID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15, or a subsequence thereof; (b)isolating a nucleic acid from the environmental sample or treating theenvironmental sample such that nucleic acid in the sample is accessiblefor hybridization to the amplification primer pair; and, (c) combiningthe nucleic acid of step (b) with the amplification primer pair of step(a) and amplifying nucleic acid from the environmental sample, therebyisolating or recovering a nucleic acid encoding a polypeptide having anamylase activity from an environmental sample. One or each member of theamplification primer sequence pair can comprise an oligonucleotidecomprising at least about 10 to 50 consecutive bases of a sequence asset forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or a subsequencethereof.

The invention provides methods for isolating or recovering a nucleicacid encoding a polypeptide having an amylase activity from anenvironmental sample comprising the steps of: (a) providing apolynucleotide probe comprising a nucleic acid of the invention or asubsequence thereof, including a nucleic acid having at least 90% ormore sequence identity to SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:9; (b)isolating a nucleic acid from the environmental sample or treating theenvironmental sample such that nucleic acid in the sample is accessiblefor hybridization to a polynucleotide probe of step (a); (c) combiningthe isolated nucleic acid or the treated environmental sample of step(b) with the polynucleotide probe of step (a); and (d) isolating anucleic acid that specifically hybridizes with the polynucleotide probeof step (a), thereby isolating or recovering a nucleic acid encoding apolypeptide having an amylase activity from an environmental sample. Theenvironmental sample can comprise a water sample, a liquid sample, asoil sample, an air sample or a biological sample. In one aspect, thebiological sample can be derived from a bacterial cell, a protozoancell, an insect cell, a yeast cell, a plant cell, a fungal cell or amammalian cell.

The invention provides methods of generating a variant of a nucleic acidencoding a polypeptide having an amylase activity comprising the stepsof: (a) providing a template nucleic acid comprising a nucleic acid ofthe invention; and (b) modifying, deleting or adding one or morenucleotides in the template sequence, or a combination thereof, togenerate a variant of the template nucleic acid. In one aspect, themethod can further comprise expressing the variant nucleic acid togenerate a variant amylase polypeptide. The modifications, additions ordeletions can be introduced by a method comprising error-prone PCR,shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexualPCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursiveensemble mutagenesis, exponential ensemble mutagenesis, site-specificmutagenesis, gene reassembly, gene site saturated mutagenesis (GSSM),synthetic ligation reassembly (SLR) or a combination thereof. In anotheraspect, the modifications, additions or deletions are introduced by amethod comprising recombination, recursive sequence recombination,phosphothioate-modified DNA mutagenesis, uracil-containing templatemutagenesis, gapped duplex mutagenesis, point mismatch repairmutagenesis, repair-deficient host strain mutagenesis, chemicalmutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acidmultimer creation and a combination thereof.

In one aspect, the method can be iteratively repeated until an amylasehaving an altered or different activity or an altered or differentstability from that of a polypeptide encoded by the template nucleicacid is produced. In one aspect, the variant amylase polypeptide isthermotolerant, and retains some activity after being exposed to anelevated temperature. In another aspect, the variant amylase polypeptidehas increased glycosylation as compared to the amylase encoded by atemplate nucleic acid. Alternatively, the variant amylase polypeptidehas an amylase activity under a high temperature, wherein the amylaseencoded by the template nucleic acid is not active under the hightemperature. In one aspect, the method can be iteratively repeated untilan amylase coding sequence having an altered codon usage from that ofthe template nucleic acid is produced. In another aspect, the method canbe iteratively repeated until an amylase gene having higher or lowerlevel of message expression or stability from that of the templatenucleic acid is produced.

The invention provides methods for modifying codons in a nucleic acidencoding a polypeptide having an amylase activity to increase itsexpression in a host cell, the method comprising the following steps:(a) providing a nucleic acid of the invention encoding a polypeptidehaving an amylase activity; and, (b) identifying a non-preferred or aless preferred codon in the nucleic acid of step (a) and replacing itwith a preferred or neutrally used codon encoding the same amino acid asthe replaced codon, wherein a preferred codon is a codonover-represented in coding sequences in genes in the host cell and anon-preferred or less preferred codon is a codon under-represented incoding sequences in genes in the host cell, thereby modifying thenucleic acid to increase its expression in a host cell. The inventionprovides methods for modifying codons in a nucleic acid of the inventionencoding a polypeptide having an amylase activity; the method comprisingthe following steps: (a) providing a nucleic acid of the invention; and,(b) identifying a codon in the nucleic acid of step (a) and replacing itwith a different codon encoding the same amino acid as the replacedcodon, thereby modifying codons in a nucleic acid encoding an amylase.

The invention provides methods for modifying codons in a nucleic acidencoding a polypeptide having an amylase activity to increase itsexpression in a host cell, the method comprising the following steps:(a) providing a nucleic acid of the invention encoding an amylasepolypeptide; and, (b) identifying a non-preferred or a less preferredcodon in the nucleic acid of step (a) and replacing it with a preferredor neutrally used codon encoding the same amino acid as the replacedcodon, wherein a preferred codon is a codon over-represented in codingsequences in genes in the host cell and a non-preferred or lesspreferred codon is a codon under-represented in coding sequences ingenes in the host cell, thereby modifying the nucleic acid to increaseits expression in a host cell.

The invention provides methods for modifying a codon in a nucleic acidencoding a polypeptide having an amylase activity to decrease itsexpression in a host cell, the method comprising the following steps:(a) providing a nucleic acid of the invention; and (b) identifying atleast one preferred codon in the nucleic acid of step (a) and replacingit with a non-preferred or less preferred codon encoding the same aminoacid as the replaced codon, wherein a preferred codon is a codonover-represented in coding sequences in genes in a host cell and anon-preferred or less preferred codon is a codon under-represented incoding sequences in genes in the host cell, thereby modifying thenucleic acid to decrease its expression in a host cell. In one aspect,the host cell can be a bacterial cell, a fungal cell, an insect cell, ayeast cell, a plant cell or a mammalian cell.

The invention provides methods for producing a library of nucleic acidsencoding a plurality of modified amylase active sites or substratebinding sites, wherein the modified active sites or substrate bindingsites are derived from a first nucleic acid comprising a sequenceencoding a first active site or a first substrate binding site themethod comprising the following steps: (a) providing a first nucleicacid encoding a first active site or first substrate binding site,wherein the first nucleic acid sequence comprises a sequence thathybridizes under stringent conditions to a sequence as set forth in SEQID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15, or a subsequence thereof, and thenucleic acid encodes an amylase active site or an amylase substratebinding site; (b) providing a set of mutagenic oligonucleotides thatencode naturally-occurring amino acid variants at a plurality oftargeted codons in the first nucleic acid; and, (c) using the set ofmutagenic oligonucleotides to generate a set of active site-encoding orsubstrate binding site-encoding variant nucleic acids encoding a rangeof amino acid variations at each amino acid codon that was mutagenized,thereby producing a library of nucleic acids encoding a plurality ofmodified amylase active sites or substrate binding sites. In one aspect,the method comprises mutagenizing the first nucleic acid of step (a) bya method comprising an optimized directed evolution system, genesite-saturation mutagenesis (GSSM), synthetic ligation reassembly (SLR),error-prone PCR, shuffling, oligonucleotide-directed mutagenesis,assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassettemutagenesis, recursive ensemble mutagenesis, exponential ensemblemutagenesis, site-specific mutagenesis, gene reassembly, gene sitesaturated mutagenesis (GSSM), synthetic ligation reassembly (SLR) and acombination thereof. In another aspect, the method comprisesmutagenizing the first nucleic acid of step (a) or variants by a methodcomprising recombination, recursive sequence recombination,phosphothioate-modified DNA mutagenesis, uracil-containing templatemutagenesis, gapped duplex mutagenesis, point mismatch repairmutagenesis, repair-deficient host strain mutagenesis, chemicalmutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acidmultimer creation and a combination thereof.

The invention provides methods for making a small molecule comprisingthe following steps: (a) providing a plurality of biosynthetic enzymescapable of synthesizing or modifying a small molecule, wherein one ofthe enzymes comprises an amylase enzyme encoded by a nucleic acid of theinvention; (b) providing a substrate for at least one of the enzymes ofstep (a); and (c) reacting the substrate of step (b) with the enzymesunder conditions that facilitate a plurality of biocatalytic reactionsto generate a small molecule by a series of biocatalytic reactions.

The invention provides methods for modifying a small molecule comprisingthe following steps: (a) providing an amylase enzyme, wherein the enzymecomprises a polypeptide of the invention, or, a polypeptide encoded by anucleic acid of the invention, or a subsequence thereof; (b) providing asmall molecule; and (c) reacting the enzyme of step (a) with the smallmolecule of step (b) under conditions that facilitate an enzymaticreaction catalyzed by the amylase enzyme, thereby modifying a smallmolecule by an amylase enzymatic reaction. In one aspect, the method cancomprise a plurality of small molecule substrates for the enzyme of step(a), thereby generating a library of modified small molecules producedby at least one enzymatic reaction catalyzed by the amylase enzyme. Inone aspect, the method can comprise a plurality of additional enzymesunder conditions that facilitate a plurality of biocatalytic reactionsby the enzymes to form a library of modified small molecules produced bythe plurality of enzymatic reactions. In another aspect, the method canfurther comprise the step of testing the library to determine if aparticular modified small molecule which exhibits a desired activity ispresent within the library. The step of testing the library can furthercomprise the steps of systematically eliminating all but one of thebiocatalytic reactions used to produce a portion of the plurality of themodified small molecules within the library by testing the portion ofthe modified small molecule for the presence or absence of theparticular modified small molecule with a desired activity, andidentifying at least one specific biocatalytic reaction that producesthe particular modified small molecule of desired activity.

The invention provides methods for determining a functional fragment ofan amylase enzyme comprising the steps of: (a) providing an amylaseenzyme, wherein the enzyme comprises a polypeptide of the invention, ora polypeptide encoded by a nucleic acid of the invention, or asubsequence thereof; and (b) deleting a plurality of amino acid residuesfrom the sequence of step (a) and testing the remaining subsequence foran amylase activity, thereby determining a functional fragment of anamylase enzyme. In one aspect, the amylase activity is measured byproviding an amylase substrate and detecting a decrease in the amount ofthe substrate or an increase in the amount of a reaction product.

The invention provides methods for whole cell engineering of new ormodified phenotypes by using real-time metabolic flux analysis, themethod comprising the following steps: (a) making a modified cell bymodifying the genetic composition of a cell, wherein the geneticcomposition is modified by addition to the cell of a nucleic acid of theinvention; (b) culturing the modified cell to generate a plurality ofmodified cells; (c) measuring at least one metabolic parameter of thecell by monitoring the cell culture of step (b) in real time; and, (d)analyzing the data of step (c) to determine if the measured parameterdiffers from a comparable measurement in an unmodified cell undersimilar conditions, thereby identifying an engineered phenotype in thecell using real-time metabolic flux analysis. In one aspect, the geneticcomposition of the cell can be modified by a method comprising deletionof a sequence or modification of a sequence in the cell, or, knockingout the expression of a gene. In one aspect, the method can furthercomprise selecting a cell comprising a newly engineered phenotype. Inanother aspect, the method can comprise culturing the selected cell,thereby generating a new cell strain comprising a newly engineeredphenotype.

The invention provides methods for hydrolyzing a starch comprising thefollowing steps: (a) providing a polypeptide having an amylase activity,wherein the polypeptide comprises a polypeptide of the invention, or apolypeptide encoded by a nucleic acid of the invention, or a subsequencethereof; (b) providing a composition comprising a starch; and (c)contacting the polypeptide of step (a) with the composition of step (b)under conditions wherein the polypeptide hydrolyzes the starch. In oneaspect, the composition comprising starch that comprises anα-1,4-glucosidic bond or an α-1,6-glucosidic bond.

The invention provides methods for liquefying or removing a starch froma composition comprising the following steps: (a) providing apolypeptide having an amylase activity, wherein the polypeptidecomprises a polypeptide of the invention, or a polypeptide encoded by anucleic acid of the invention; (b) providing a composition comprising astarch; and (c) contacting the polypeptide of step (a) with thecomposition of step (b) under conditions wherein the polypeptide removesor liquefies the starch.

The invention provides methods of increasing thermotolerance orthermostability of an amylase polypeptide, the method comprisingglycosylating an amylase polypeptide, wherein the polypeptide comprisesat least thirty contiguous amino acids of a polypeptide of theinvention; or a polypeptide encoded by a nucleic acid sequence of theinvention, thereby increasing the thermotolerance or thermostability ofthe amylase polypeptide. In one aspect, the amylase specific activitycan be thermostable or thermotolerant at a temperature in the range fromgreater than about 37° C. to about 95° C.

The invention provides methods for overexpressing a recombinant amylasepolypeptide in a cell comprising expressing a vector comprising anucleic acid comprising a nucleic acid of the invention or a nucleicacid sequence of the invention, wherein the sequence identities aredetermined by analysis with a sequence comparison algorithm or by visualinspection, wherein overexpression is effected by use of a high activitypromoter, a dicistronic vector or by gene amplification of the vector.

The invention provides detergent compositions comprising a polypeptideof the invention or a polypeptide encoded by a nucleic acid of theinvention, wherein the polypeptide comprises an amylase activity. In oneaspect, the amylase can be a nonsurface-active amylase. In anotheraspect, the amylase can be a surface-active amylase.

The invention provides methods for washing an object comprising thefollowing steps: (a) providing a composition comprising a polypeptidehaving an amylase activity, wherein the polypeptide comprises: apolypeptide of the invention or a polypeptide encoded by a nucleic acidof the invention; (b) providing an object; and (c) contacting thepolypeptide of step (a) and the object of step (b) under conditionswherein the composition can wash the object.

The invention provides methods for hydrolyzing starch, e.g., in a feedor a food prior to consumption by an animal, comprising the followingsteps: (a) obtaining a composition, e.g., a feed material, comprising astarch, wherein the polypeptide comprises: a polypeptide of theinvention or a polypeptide encoded by a nucleic acid of the invention;and (b) adding the polypeptide of step (a) to the composition, e.g., thefeed or food material, in an amount sufficient for a sufficient timeperiod to cause hydrolysis of the starch, thereby hydrolyzing thestarch. In one aspect, the food or feed comprises rice, corn, barley,wheat, legumes, or potato.

The invention provides methods for textile desizing comprising thefollowing steps: (a) providing a polypeptide having an amylase activity,wherein the polypeptide comprises a polypeptide of the invention or apolypeptide encoded by a nucleic acid of the invention; (b) providing afabric; and (c) contacting the polypeptide of step (a) and the fabric ofstep (b) under conditions wherein the amylase can desize the fabric.

The invention provides methods for deinking of paper or fiberscomprising the following steps: (a) providing a polypeptide having anamylase activity, wherein the polypeptide comprises a polypeptide of theinvention or a polypeptide encoded by a nucleic acid of the invention;(b) providing a composition comprising paper or fiber; and (c)contacting the polypeptide of step (a) and the composition of step (b)under conditions wherein the polypeptide can deink the paper or fiber.

The invention provides methods for treatment of lignocellulosic fiberscomprising the following steps: (a) providing a polypeptide having anamylase activity, wherein the polypeptide comprises a polypeptide of theinvention or a polypeptide encoded by a nucleic acid of the invention;(b) providing a lignocellulosic fiber; and (c) contacting thepolypeptide of step (a) and the fiber of step (b) under conditionswherein the polypeptide can treat the fiber thereby improving the fiberproperties.

The invention provides methods for producing a high-maltose or ahigh-glucose syrup comprising the following steps: (a) providing apolypeptide having an amylase activity, wherein the polypeptidecomprises a polypeptide of the invention or a polypeptide encoded by anucleic acid of the invention; (b) providing a composition comprising astarch; and (c) contacting the polypeptide of step (a) and the fabric ofstep (b) under conditions wherein the polypeptide of step (a) canliquefy the composition of step (b) thereby producing a soluble starchhydrolysate and saccharify the soluble starch hydrolysate therebyproducing the syrup. In one aspect, the starch can be from rice, corn,barley, wheat, legumes, potato, or sweet potato.

The invention provides methods for improving the flow of thestarch-containing production fluids comprising the following steps: (a)providing a polypeptide having an amylase activity, wherein thepolypeptide comprises a polypeptide of the invention or a polypeptideencoded by a nucleic acid of the invention; (b) providing productionfluid; and (c) contacting the polypeptide of step (a) and the productionfluid of step (b) under conditions wherein the amylase can hydrolyze thestarch in the production fluid thereby improving its flow by decreasingits density. In one aspect, the production fluid can be from asubterranean formation.

The invention provides anti-staling compositions comprising apolypeptide of the invention or a polypeptide encoded by a nucleic acidof the invention. The invention provides methods for preventing stalingof the baked products comprising the following steps: (a) providing apolypeptide having an amylase activity, wherein the polypeptidecomprises a polypeptide of the invention or a polypeptide encoded by anucleic acid of the invention; (b) providing a composition containingstarch used for baking; (c) combining the polypeptide of step (a) withthe composition of the step (b) under conditions wherein the polypeptidecan hydrolyze the starch in the composition used for baking therebypreventing staling of the baked product. In one aspect, the bakedproduct can be bread.

The invention provides methods for using amylase in brewing or alcoholproduction comprising the following steps: (a) providing a polypeptidehaving an amylase activity, wherein the polypeptide comprises apolypeptide of the invention or a polypeptide encoded by a nucleic acidof the invention; (b) providing a composition containing starch and usedfor brewing or in alcohol production; (c) combining the polypeptide ofstep (a) with the composition of the step (b) under conditions whereinthe polypeptide can hydrolyze the starch in the composition used forbrewing or in alcohol production. In one aspect, the compositioncontaining starch can be beer.

The invention provides methods of making a transgenic plant comprisingthe following steps: (a) introducing a heterologous nucleic acidsequence into the cell, wherein the heterologous nucleic sequencecomprises a nucleic acid sequence of the invention, thereby producing atransformed plant cell; and (b) producing a transgenic plant from thetransformed cell. In one aspect, the step (a) can further compriseintroducing the heterologous nucleic acid sequence by electroporation ormicroinjection of plant cell protoplasts. In another aspect, the step(a) can further comprise introducing the heterologous nucleic acidsequence directly to plant tissue by DNA particle bombardment.Alternatively, the step (a) can further comprise introducing theheterologous nucleic acid sequence into the plant cell DNA using anAgrobacterium tumefaciens host. In one aspect, the plant cell can be apotato, corn, rice, wheat, tobacco, or barley cell.

The invention provides methods of expressing a heterologous nucleic acidsequence in a plant cell comprising the following steps: (a)transforming the plant cell with a heterologous nucleic acid sequenceoperably linked to a promoter, wherein the heterologous nucleic sequencecomprises a nucleic acid of the invention; (b) growing the plant underconditions wherein the heterologous nucleic acids sequence is expressedin the plant cell.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

All publications, patents, patent applications, GenBank sequences andATCC deposits, cited herein are hereby expressly incorporated byreference for all purposes.

DESCRIPTION OF DRAWINGS

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

FIG. 1 is a block diagram of a computer system.

FIG. 2 is a flow diagram illustrating one aspect of a process forcomparing a new nucleotide or protein sequence with a database ofsequences in order to determine the homology levels between the newsequence and the sequences in the database.

FIG. 3 is a flow diagram illustrating one aspect of a process in acomputer for determining whether two sequences are homologous.

FIG. 4 is a flow diagram illustrating one aspect of an identifierprocess 300 for detecting the presence of a feature in a sequence.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

The invention provides amylase enzymes, polynucleotides encoding theenzymes, methods of making and using these polynucleotides andpolypeptides. The invention is directed to novel polypeptides having anamylase activity, nucleic acids encoding them and antibodies that bindto them. The polypeptides of the invention can be used in a variety ofdiagnostic, therapeutic, and industrial contexts. The polypeptides ofthe invention can be used as, e.g., an additive for a detergent, forprocessing foods and for chemical synthesis utilizing a reversereaction. Additionally, the polypeptides of the invention can be used infabric treatment, alcohol production, and as additives to food or animalfeed.

In one aspect, the amylases of the invention are active at a high and/orat a low temperature, or, over a wide range of temperature. For example,they can be active in the temperatures ranging between 20° C. to 90° C.,between 30° C. to 80° C., or between 40° C. to 70° C. The invention alsoprovides amylases that have activity at alkaline pHs or at acidic pHs,e.g., low water acidity. In alternative aspects, the amylases of theinvention can have activity in acidic pHs as low as pH 5.0, pH 4.5, pH4.0, and pH 3.5. In alternative aspects, the amylases of the inventioncan have activity in alkaline pHs as high as pH 9.5, pH 10, pH 10.5, andpH 11. In one aspect, the amylases of the invention are active in thetemperature range of between about 40° C. to about 70° C. underconditions of low water activity (low water content).

The invention also provides methods for further modifying the exemplaryamylases of the invention to generate proteins with desirableproperties. For example, amylases generated by the methods of theinvention can have altered enzymatic activity, thermal stability,pH/activity profile, pH/stability profile (such as increased stabilityat low, e.g. pH<6 or pH<5, or high, e.g. pH>9, pH values), stabilitytowards oxidation, Ca²⁺ dependency, specific activity and the like. Theinvention provides for altering any property of interest. For instance,the alteration may result in a variant which, as compared to a parentenzyme, has altered enzymatic activity, or, pH or temperature activityprofiles.

DEFINITIONS

The term “amylase” includes all polypeptides, e.g., enzymes orantibodies, that catalyze the hydrolysis of starches. For example, theterm amylase, and the amylases of the invention, include polypeptideshaving glucoamylase activity, such as the ability to catalyze thehydrolysis of glucose polymers, e.g., glucose polymers linked by α-1,4-and/or α-1,6-glucosidic bonds. In one aspect, the polypeptides of theinvention have glucoamylase activity, hydrolyzing internalα-1,4-glucosidic linkages to yield smaller molecular weightmalto-dextrins. An amylase activity of the invention also includesα-amylase activity, including the ability to hydrolyze internalalpha-1,4-glucosidic linkages in starch to produce smaller molecularweight malto-dextrins. In one aspect, the α-amylase activity of theinvention includes hydrolyzing internal alpha-1,4-glucosidic linkages instarch at random. An amylase activity of the invention also includesglucan 1,4-α-glucosidase activity, or, 1,4-α-D-glucan glucohydrolase,commonly called glucoamylase but also called amyloglucosidase andγ-amylase that, in one aspect, releases β-D-glucose from 1,4-α-, 1,6-α-and 1,3-α-linked glucans. An amylase activity of the invention alsoincludes exo-amylase activity. An amylase activity of the invention alsoincludes hydrolyzing starch at high temperatures, low temperatures,alkaline pHs and at acidic pHs.

An “amylase variant” comprises an amino acid sequence which is derivedfrom the amino acid sequence of a “precursor amylase”. The precursoramylase can include naturally-occurring amylases and recombinantamylases. The amino acid sequence of the amylase variant can be“derived” from the precursor amylase amino acid sequence by thesubstitution, deletion or insertion of one or more amino acids of theprecursor amino acid sequence. Such modification can be of the“precursor DNA sequence” which encodes the amino acid sequence of theprecursor amylase rather than manipulation of the precursor amylaseenzyme per se. Suitable methods for manipulation of the precursor DNAsequence include methods disclosed herein, as well as methods known tothose skilled in the art.

The term “antibody” includes a peptide or polypeptide derived from,modeled after or substantially encoded by an immunoglobulin gene orimmunoglobulin genes, or fragments thereof, capable of specificallybinding an antigen or epitope, see, e.g. Fundamental Immunology, ThirdEdition, W. E. Paul, ed., Raven Press, N.Y. (1993); Wilson (1994) J.Immunol. Methods 175:267-273; Yarmush (1992) J. Biochem. Biophys.Methods 25:85-97. The term antibody includes antigen-binding portions,i.e., “antigen binding sites,” (e.g., fragments, subsequences,complementarity determining regions (CDRs)) that retain capacity to bindantigen, including (i) a Fab fragment, a monovalent fragment consistingof the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting of the VH and CH1domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341:544-546), which consists of a VH domain; and (vi) an isolatedcomplementarity determining region (CDR). Single chain antibodies arealso included by reference in the term “antibody.”

The terms “array” or “microarray” or “biochip” or “chip” as used hereinis a plurality of target elements, each target element comprising adefined amount of one or more polypeptides (including antibodies) ornucleic acids immobilized onto a defined area of a substrate surface, asdiscussed in further detail, below.

As used herein, the terms “computer,” “computer program” and “processor”are used in their broadest general contexts and incorporate all suchdevices, as described in detail, below. A “coding sequence of” or a“sequence encodes” a particular polypeptide or protein, is a nucleicacid sequence which is transcribed and translated into a polypeptide orprotein when placed under the control of appropriate regulatorysequences.

The term “expression cassette” as used herein refers to a nucleotidesequence which is capable of affecting expression of a structural gene(i.e., a protein coding sequence, such as an amylase of the invention)in a host compatible with such sequences. Expression cassettes includeat least a promoter operably linked with the polypeptide codingsequence; and, optionally, with other sequences, e.g., transcriptiontermination signals. Additional factors necessary or helpful ineffecting expression may also be used, e.g., enhancers. Thus, expressioncassettes also include plasmids, expression vectors, recombinantviruses, any form of recombinant “naked DNA” vector, and the like.

“Operably linked” as used herein refers to a functional relationshipbetween two or more nucleic acid (e.g., DNA) segments. Typically, itrefers to the functional relationship of transcriptional regulatorysequence to a transcribed sequence. For example, a promoter is operablylinked to a coding sequence, such as a nucleic acid of the invention, ifit stimulates or modulates the transcription of the coding sequence inan appropriate host cell or other expression system. Generally, promotertranscriptional regulatory sequences that are operably linked to atranscribed sequence are physically contiguous to the transcribedsequence, i.e., they are cis-acting. However, some transcriptionalregulatory sequences, such as enhancers, need not be physicallycontiguous or located in close proximity to the coding sequences whosetranscription they enhance.

A “vector” comprises a nucleic acid which can infect, transfect,transiently or permanently transduce a cell. It will be recognized thata vector can be a naked nucleic acid, or a nucleic acid complexed withprotein or lipid. The vector optionally comprises viral or bacterialnucleic acids and/or proteins, and/or membranes (e.g., a cell membrane,a viral lipid envelope, etc.). Vectors include, but are not limited toreplicons (e.g., RNA replicons, bacteriophages) to which fragments ofDNA may be attached and become replicated. Vectors thus include, but arenot limited to RNA, autonomous self-replicating circular or linear DNAor RNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Pat. No.5,217,879), and include both the expression and non-expression plasmids.Where a recombinant microorganism or cell culture is described ashosting an “expression vector” this includes both extra-chromosomalcircular and linear DNA and DNA that has been incorporated into the hostchromosome(s). Where a vector is being maintained by a host cell, thevector may either be stably replicated by the cells during mitosis as anautonomous structure, or is incorporated within the host's genome.

As used herein, the term “promoter” includes all sequences capable ofdriving transcription of a coding sequence in a cell, e.g., a plantcell. Thus, promoters used in the constructs of the invention includecis-acting transcriptional control elements and regulatory sequencesthat are involved in regulating or modulating the timing and/or rate oftranscription of a gene. For example, a promoter can be a cis-actingtranscriptional control element, including an enhancer, a promoter, atranscription terminator, an origin of replication, a chromosomalintegration sequence, 5′ and 3′ untranslated regions, or an intronicsequence, which are involved in transcriptional regulation. Thesecis-acting sequences typically interact with proteins or otherbiomolecules to carry out (turn on/off, regulate, modulate, etc.)transcription. “Constitutive” promoters are those that drive expressioncontinuously under most environmental conditions and states ofdevelopment or cell differentiation. “Inducible” or “regulatable”promoters direct expression of the nucleic acid of the invention underthe influence of environmental conditions or developmental conditions.Examples of environmental conditions that may affect transcription byinducible promoters include anaerobic conditions, elevated temperature,drought, or the presence of light.

“Tissue-specific” promoters are transcriptional control elements thatare only active in particular cells or tissues or organs, e.g., inplants or animals. Tissue-specific regulation may be achieved by certainintrinsic factors which ensure that genes encoding proteins specific toa given tissue are expressed. Such factors are known to exist in mammalsand plants so as to allow for specific tissues to develop.

The term “plant” includes whole plants, plant parts (e.g., leaves,stems, flowers, roots, etc.), plant protoplasts, seeds and plant cellsand progeny of same. The class of plants which can be used in the methodof the invention is generally as broad as the class of higher plantsamenable to transformation techniques, including angiosperms(monocotyledonous and dicotyledonous plants), as well as gymnosperms. Itincludes plants of a variety of ploidy levels, including polyploid,diploid, haploid and hemizygous states. As used herein, the term“transgenic plant” includes plants or plant cells into which aheterologous nucleic acid sequence has been inserted, e.g., the nucleicacids and various recombinant constructs (e.g., expression cassettes) ofthe invention.

“Plasmids” can be commercially available, publicly available on anunrestricted basis, or can be constructed from available plasmids inaccord with published procedures. Equivalent plasmids to those describedherein are known in the art and will be apparent to the ordinarilyskilled artisan.

The term “gene” includes a nucleic acid sequence comprising a segment ofDNA involved in producing a transcription product (e.g., a message),which in turn is translated to produce a polypeptide chain, or regulatesgene transcription, reproduction or stability. Genes can include regionspreceding and following the coding region, such as leader and trailer,promoters and enhancers, as well as, where applicable, interveningsequences (introns) between individual coding segments (exons).

The phrases “nucleic acid” or “nucleic acid sequence” includesoligonucleotide, nucleotide, polynucleotide, or to a fragment of any ofthese, to DNA or RNA (e.g., mRNA, rRNA, tRNA) of genomic or syntheticorigin which may be single-stranded or double-stranded and may representa sense or antisense strand, to peptide nucleic acid (PNA), or to anyDNA-like or RNA-like material, natural or synthetic in origin,including, e.g., iRNA, ribonucleoproteins (e.g., iRNPs). The termencompasses nucleic acids, i.e., oligonucleotides, containing knownanalogues of natural nucleotides. The term also encompassesnucleic-acid-like structures with synthetic backbones, see e.g., Mata(1997) Toxicol. Appl. Pharmacol. 144:189-197; Strauss-Soukup (1997)Biochemistry 36:8692-8698; Samstag (1996) Antisense Nucleic Acid DrugDev 6:153-156.

“Amino acid” or “amino acid sequence” include an oligopeptide, peptide,polypeptide, or protein sequence, or to a fragment, portion, or subunitof any of these, and to naturally occurring or synthetic molecules. Theterms “polypeptide” and “protein” include amino acids joined to eachother by peptide bonds or modified peptide bonds, i.e., peptideisosteres, and may contain modified amino acids other than the 20gene-encoded amino acids. The term “polypeptide” also includes peptidesand polypeptide fragments, motifs and the like. The term also includesglycosylated polypeptides. The peptides and polypeptides of theinvention also include all “mimetic” and “peptidomimetic” forms, asdescribed in further detail, below.

The term “isolated” includes a material removed from its originalenvironment, e.g., the natural environment if it is naturally occurring.For example, a naturally occurring polynucleotide or polypeptide presentin a living animal is not isolated, but the same polynucleotide orpolypeptide, separated from some or all of the coexisting materials inthe natural system, is isolated. Such polynucleotides could be part of avector and/or such polynucleotides or polypeptides could be part of acomposition, and still be isolated in that such vector or composition isnot part of its natural environment. As used herein, an isolatedmaterial or composition can also be a “purified” composition, i.e., itdoes not require absolute purity; rather, it is intended as a relativedefinition. Individual nucleic acids obtained from a library can beconventionally purified to electrophoretic homogeneity. In alternativeaspects, the invention provides nucleic acids which have been purifiedfrom genomic DNA or from other sequences in a library or otherenvironment by at least one, two, three, four, five or more orders ofmagnitude.

As used herein, the term “recombinant” includes nucleic acids adjacentto a “backbone” nucleic acid to which it is not adjacent in its naturalenvironment. In one aspect, nucleic acids represent 5% or more of thenumber of nucleic acid inserts in a population of nucleic acid “backbonemolecules.” “Backbone molecules” according to the invention includenucleic acids such as expression vectors, self-replicating nucleicacids, viruses, integrating nucleic acids, and other vectors or nucleicacids used to maintain or manipulate a nucleic acid insert of interest.In one aspect, the enriched nucleic acids represent 10%, 15%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or more of the number of nucleicacid inserts in the population of recombinant backbone molecules.“Recombinant” polypeptides or proteins refer to polypeptides or proteinsproduced by recombinant DNA techniques; e.g., produced from cellstransformed by an exogenous DNA construct encoding the desiredpolypeptide or protein. “Synthetic” polypeptides or protein are thoseprepared by chemical synthesis, as described in further detail, below.

A promoter sequence can be “operably linked to” a coding sequence whenRNA polymerase which initiates transcription at the promoter willtranscribe the coding sequence into mRNA, as discussed further, below.

“Oligonucleotide” includes either a single stranded polydeoxynucleotideor two complementary polydeoxynucleotide strands which may be chemicallysynthesized. Such synthetic oligonucleotides have no 5′ phosphate andthus will not ligate to another oligonucleotide without adding aphosphate with an ATP in the presence of a kinase. A syntheticoligonucleotide can ligate to a fragment that has not beendephosphorylated.

The phrase “substantially identical” in the context of two nucleic acidsor polypeptides, can refer to two or more sequences that have, e.g., atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%or more nucleotide or amino acid residue (sequence) identity, whencompared and aligned for maximum correspondence, as measured using oneany known sequence comparison algorithm, as discussed in detail below,or by visual inspection. In alternative aspects, the invention providesnucleic acid and polypeptide sequences having substantial identity to anexemplary sequence of the invention, e.g., SEQ ID NO:1, SEQ ID NO:3, SEQID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:15, over a region of at least about 10, 20, 30, 40, 50, 100, 150,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1000 or more residues, or a region ranging from between about50 residues to the full length of the nucleic acid or polypeptide.Nucleic acid sequences of the invention can be substantially identicalover the entire length of a polypeptide coding region.

A “substantially identical” amino acid sequence also can include asequence that differs from a reference sequence by one or moreconservative or non-conservative amino acid substitutions, deletions, orinsertions, particularly when such a substitution occurs at a site thatis not the active site of the molecule, and provided that thepolypeptide essentially retains its functional properties. Aconservative amino acid substitution, for example, substitutes one aminoacid for another of the same class (e.g., substitution of onehydrophobic amino acid, such as isoleucine, valine, leucine, ormethionine, for another, or substitution of one polar amino acid foranother, such as substitution of arginine for lysine, glutamic acid foraspartic acid or glutamine for asparagine). One or more amino acids canbe deleted, for example, from an amylase, resulting in modification ofthe structure of the polypeptide, without significantly altering itsbiological activity. For example, amino- or carboxyl-terminal aminoacids that are not required for amylase activity can be removed.

“Hybridization” includes the process by which a nucleic acid strandjoins with a complementary strand through base pairing. Hybridizationreactions can be sensitive and selective so that a particular sequenceof interest can be identified even in samples in which it is present atlow concentrations. Stringent conditions can be defined by, for example,the concentrations of salt or formamide in the prehybridization andhybridization solutions, or by the hybridization temperature, and arewell known in the art. For example, stringency can be increased byreducing the concentration of salt, increasing the concentration offormamide, or raising the hybridization temperature, altering the timeof hybridization, as described in detail, below. In alternative aspects,nucleic acids of the invention are defined by their ability to hybridizeunder various stringency conditions (e.g., high, medium, and low), asset forth herein.

“Variant” includes polynucleotides or polypeptides of the inventionmodified at one or more base pairs, codons, introns, exons, or aminoacid residues (respectively) yet still retain the biological activity ofan amylase of the invention. Variants can be produced by any number ofmeans included methods such as, for example, error-prone PCR, shuffling,oligonucleotide-directed mutagenesis, assembly PCR, sexual PCRmutagenesis, in vivo mutagenesis, cassette mutagenesis, recursiveensemble mutagenesis, exponential ensemble mutagenesis, site-specificmutagenesis, gene reassembly, GSSM and any combination thereof.Techniques for producing variant amylase having activity at a pH ortemperature, for example, that is different from a wild-type amylase,are included herein.

The term “saturation mutagenesis” or “GSSM” includes a method that usesdegenerate oligonucleotide primers to introduce point mutations into apolynucleotide, as described in detail, below.

The term “optimized directed evolution system” or “optimized directedevolution” includes a method for reassembling fragments of relatednucleic acid sequences, e.g., related genes, and explained in detail,below.

The term “synthetic ligation reassembly” or “SLR” includes a method ofligating oligonucleotide fragments in a non-stochastic fashion, andexplained in detail, below.

Generating and Manipulating Nucleic Acids

The invention provides nucleic acids, including expression cassettessuch as expression vectors, encoding the polypeptides of the invention.The invention also includes methods for discovering new amylasesequences using the nucleic acids of the invention. The invention alsoincludes methods for inhibiting the expression of amylase genes,transcripts and polypeptides using the nucleic acids of the invention.Also provided are methods for modifying the nucleic acids of theinvention by, e.g., synthetic ligation reassembly, optimized directedevolution system and/or saturation mutagenesis. The nucleic acids of theinvention can be made, isolated and/or manipulated by, e.g., cloning andexpression of cDNA libraries, amplification of message or genomic DNA byPCR, and the like. In practicing the methods of the invention,homologous genes can be modified by manipulating a template nucleicacid, as described herein. The invention can be practiced in conjunctionwith any method or protocol or device known in the art, which are welldescribed in the scientific and patent literature.

General Techniques

The nucleic acids used to practice this invention, whether RNA, iRNA,antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybridsthereof, may be isolated from a variety of sources, geneticallyengineered, amplified, and/or expressed/generated recombinantly.Recombinant polypeptides generated from these nucleic acids can beindividually isolated or cloned and tested for a desired activity. Anyrecombinant expression system can be used, including bacterial,mammalian, yeast, insect or plant cell expression systems.

Alternatively, these nucleic acids can be synthesized in vitro bywell-known chemical synthesis techniques, as described in, e.g., Adams(1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res.25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers(1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90;Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett.22:1859; U.S. Pat. No. 4,458,066.

Techniques for the manipulation of nucleic acids, such as, e.g.,subcloning, labeling probes (e.g., random-primer labeling using Klenowpolymerase, nick translation, amplification), sequencing, hybridizationand the like are well described in the scientific and patent literature,see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2NDED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc.,New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULARBIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory andNucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).

Another useful means of obtaining and manipulating nucleic acids used topractice the methods of the invention is to clone from genomic samples,and, if desired, screen and re-clone inserts isolated or amplified from,e.g., genomic clones or cDNA clones. Sources of nucleic acid used in themethods of the invention include genomic or cDNA libraries contained in,e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos.5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld(1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC);bacterial artificial chromosomes (BAC); P1 artificial chromosomes, see,e.g., Woon (1998) Genomics 50:306-316; P1-derived vectors (PACs), see,e.g., Kern (1997) Biotechniques 23:120-124; cosmids, recombinantviruses, phages or plasmids.

In one aspect, a nucleic acid encoding a polypeptide of the invention isassembled in appropriate phase with a leader sequence capable ofdirecting secretion of the translated polypeptide or fragment thereof.

The invention provides fusion proteins and nucleic acids encoding them.A polypeptide of the invention can be fused to a heterologous peptide orpolypeptide, such as N-terminal identification peptides which impartdesired characteristics, such as increased stability or simplifiedpurification. Peptides and polypeptides of the invention can also besynthesized and expressed as fusion proteins with one or more additionaldomains linked thereto for, e.g., producing a more immunogenic peptide,to more readily isolate a recombinantly synthesized peptide, to identifyand isolate antibodies and antibody-expressing B cells, and the like.Detection and purification facilitating domains include, e.g., metalchelating peptides such as polyhistidine tracts and histidine-tryptophanmodules that allow purification on immobilized metals, protein A domainsthat allow purification on immobilized immunoglobulin, and the domainutilized in the FLAGS extension/affinity purification system (ImmunexCorp, Seattle Wash.). The inclusion of a cleavable linker sequences suchas Factor Xa or enterokinase (Invitrogen, San Diego Calif.) between apurification domain and the motif-comprising peptide or polypeptide tofacilitate purification. For example, an expression vector can includean epitope-encoding nucleic acid sequence linked to six histidineresidues followed by a thioredoxin and an enterokinase cleavage site(see e.g., Williams (1995) Biochemistry 34:1787-1797; Dobeli (1998)Protein Expr. Purif. 12:404-414). The histidine residues facilitatedetection and purification while the enterokinase cleavage site providesa means for purifying the epitope from the remainder of the fusionprotein. Technology pertaining to vectors encoding fusion proteins andapplication of fusion proteins are well described in the scientific andpatent literature, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53.

Transcriptional and Translational Control Sequences

The invention provides nucleic acid (e.g., DNA) sequences of theinvention operatively linked to expression (e.g., transcriptional ortranslational) control sequence(s), e.g., promoters or enhancers, todirect or modulate RNA synthesis/expression. The expression controlsequence can be in an expression vector. Exemplary bacterial promotersinclude lacI, lacZ, T3, T7, gpt, lambda PR, PL and trp. Exemplaryeukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40, LTRs from retrovirus, and mouse metallothionein I.

Promoters suitable for expressing a polypeptide in bacteria include theE. coli lac or trp promoters, the lad promoter, the lacZ promoter, theT3 promoter, the T7 promoter, the gpt promoter, the lambda PR promoter,the lambda PL promoter, promoters from operons encoding glycolyticenzymes such as 3-phosphoglycerate kinase (PGK), and the acidphosphatase promoter. Eukaryotic promoters include the CMV immediateearly promoter, the HSV thymidine kinase promoter, heat shock promoters,the early and late SV40 promoter, LTRs from retroviruses, and the mousemetallothionein-I promoter. Other promoters known to control expressionof genes in prokaryotic or eukaryotic cells or their viruses may also beused.

Tissue-Specific Plant Promoters

The invention provides expression cassettes that can be expressed in atissue-specific manner, e.g., that can express an amylase of theinvention in a tissue-specific manner. The invention also providesplants or seeds that express an amylase of the invention in atissue-specific manner. The tissue-specificity can be seed specific,stem specific, leaf specific, root specific, fruit specific and thelike.

In one aspect, a constitutive promoter such as the CaMV 35S promoter canbe used for expression in specific parts of the plant or seed orthroughout the plant. For example, for overexpression, a plant promoterfragment can be employed which will direct expression of a nucleic acidin some or all tissues of a plant, e.g., a regenerated plant. Suchpromoters are referred to herein as “constitutive” promoters and areactive under most environmental conditions and states of development orcell differentiation. Examples of constitutive promoters include thecauliflower mosaic virus (CaMV) 35S transcription initiation region, the1′- or 2′-promoter derived from T-DNA of Agrobacterium tumefaciens, andother transcription initiation regions from various plant genes known tothose of skill. Such genes include, e.g., ACT11 from Arabidopsis (Huang(1996) Plant Mol. Biol. 33:125-139); Cat3 from Arabidopsis (GenBank No.U43147, Zhong (1996) Mol. Gen. Genet. 251:196-203); the gene encodingstearoyl-acyl carrier protein desaturase from Brassica napus (GenbankNo. X74782, Solocombe (1994) Plant Physiol. 104:1167-1176); GPc1 frommaize (GenBank No. X15596; Martinez (1989) J. Mol. Biol. 208:551-565);the Gpc2 from maize (GenBank No. U45855, Manjunath (1997) Plant Mol.Biol. 33:97-112); plant promoters described in U.S. Pat. Nos. 4,962,028;5,633,440.

The invention uses tissue-specific or constitutive promoters derivedfrom viruses which can include, e.g., the tobamovirus subgenomicpromoter (Kumagai (1995) Proc. Natl. Acad. Sci. USA 92:1679-1683; therice tungro bacilliform virus (RTBV), which replicates only in phloemcells in infected rice plants, with its promoter which drives strongphloem-specific reporter gene expression; the cassava vein mosaic virus(CVMV) promoter, with highest activity in vascular elements, in leafmesophyll cells, and in root tips (Verdaguer (1996) Plant Mol. Biol.31:1129-1139).

Alternatively, the plant promoter may direct expression ofamylase-expressing nucleic acid in a specific tissue, organ or cell type(i.e. tissue-specific promoters) or may be otherwise under more preciseenvironmental or developmental control or under the control of aninducible promoter. Examples of environmental conditions that may affecttranscription include anaerobic conditions, elevated temperature, thepresence of light, or sprayed with chemicals/hormones. For example, theinvention incorporates the drought-inducible promoter of maize (Busk(1997) supra); the cold, drought, and high salt inducible promoter frompotato (Kirch (1997) Plant Mol. Biol. 33:897 909).

Tissue-specific promoters can promote transcription only within acertain time frame of developmental stage within that tissue. See, e.g.,Blazquez (1998) Plant Cell 10:791-800, characterizing the ArabidopsisLEAFY gene promoter. See also Cardon (1997) Plant J 12:367-77,describing the transcription factor SPL3, which recognizes a conservedsequence motif in the promoter region of the A. thaliana floral meristemidentity gene AP1; and Mandel (1995) Plant Molecular Biology, Vol. 29,pp 995-1004, describing the meristem promoter eIF4. Tissue specificpromoters which are active throughout the life cycle of a particulartissue can be used. In one aspect, the nucleic acids of the inventionare operably linked to a promoter active primarily only in cotton fibercells. In one aspect, the nucleic acids of the invention are operablylinked to a promoter active primarily during the stages of cotton fibercell elongation, e.g., as described by Rinehart (1996) supra. Thenucleic acids can be operably linked to the Fbl2A gene promoter to bepreferentially expressed in cotton fiber cells (Ibid). See also, John(1997) Proc. Natl. Acad. Sci. USA 89:5769-5773; John, et al., U.S. Pat.Nos. 5,608,148 and 5,602,321, describing cotton fiber-specific promotersand methods for the construction of transgenic cotton plants.Root-specific promoters may also be used to express the nucleic acids ofthe invention. Examples of root-specific promoters include the promoterfrom the alcohol dehydrogenase gene (DeLisle (1990) Int. Rev. Cytol.123:39-60). Other promoters that can be used to express the nucleicacids of the invention include, e.g., ovule-specific, embryo-specific,endosperm-specific, integument-specific, seed coat-specific promoters,or some combination thereof; a leaf-specific promoter (see, e.g., Busk(1997) Plant J. 11:1285 1295, describing a leaf-specific promoter inmaize); the ORF13 promoter from Agrobacterium rhizogenes (which exhibitshigh activity in roots, see, e.g., Hansen (1997) supra); a maize pollenspecific promoter (see, e.g., Guerrero (1990) Mol. Gen. Genet. 224:161168); a tomato promoter active during fruit ripening, senescence andabscission of leaves and, to a lesser extent, of flowers can be used(see, e.g., Blume (1997) Plant J. 12:731 746); a pistil-specificpromoter from the potato SK2 gene (see, e.g., Ficker (1997) Plant Mol.Biol. 35:425 431); the Blec4 gene from pea, which is active in epidermaltissue of vegetative and floral shoot apices of transgenic alfalfamaking it a useful tool to target the expression of foreign genes to theepidermal layer of actively growing shoots or fibers; the ovule-specificBEL1 gene (see, e.g., Reiser (1995) Cell 83:735-742, GenBank No.U39944); and/or, the promoter in Klee, U.S. Pat. No. 5,589,583,describing a plant promoter region is capable of conferring high levelsof transcription in meristematic tissue and/or rapidly dividing cells.

Alternatively, plant promoters which are inducible upon exposure toplant hormones, such as auxins, are used to express the nucleic acids ofthe invention. For example, the invention can use the auxin-responseelements E1 promoter fragment (AuxREs) in the soybean (Glycine max L.)(Liu (1997) Plant Physiol. 115:397-407); the auxin-responsiveArabidopsis GST6 promoter (also responsive to salicylic acid andhydrogen peroxide) (Chen (1996) Plant J. 10: 955-966); theauxin-inducible parC promoter from tobacco (Sakai (1996) 37:906-913); aplant biotin response element (Streit (1997) Mol. Plant. MicrobeInteract. 10:933-937); and, the promoter responsive to the stresshormone abscisic acid (Sheen (1996) Science 274:1900-1902).

The nucleic acids of the invention can also be operably linked to plantpromoters which are inducible upon exposure to chemicals reagents whichcan be applied to the plant, such as herbicides or antibiotics. Forexample, the maize In2-2 promoter, activated by benzenesulfonamideherbicide safeners, can be used (De Veylder (1997) Plant Cell Physiol.38:568-577); application of different herbicide safeners inducesdistinct gene expression patterns, including expression in the root,hydathodes, and the shoot apical meristem. Coding sequence can be underthe control of, e.g., a tetracycline-inducible promoter, e.g., asdescribed with transgenic tobacco plants containing the Avena sativa L.(oat) arginine decarboxylase gene (Masgrau (1997) Plant J. 11:465-473);or, a salicylic acid-responsive element (Stange (1997) Plant J.11:1315-1324). Using chemically- (e.g., hormone- or pesticide-) inducedpromoters, i.e., promoter responsive to a chemical which can be appliedto the transgenic plant in the field, expression of a polypeptide of theinvention can be induced at a particular stage of development of theplant. Thus, the invention also provides for transgenic plantscontaining an inducible gene encoding for polypeptides of the inventionwhose host range is limited to target plant species, such as corn, rice,barley, wheat, potato or other crops, inducible at any stage ofdevelopment of the crop.

One of skill will recognize that a tissue-specific plant promoter maydrive expression of operably linked sequences in tissues other than thetarget tissue. Thus, a tissue-specific promoter is one that drivesexpression preferentially in the target tissue or cell type, but mayalso lead to some expression in other tissues as well.

The nucleic acids of the invention can also be operably linked to plantpromoters which are inducible upon exposure to chemicals reagents. Thesereagents include, e.g., herbicides, synthetic auxins, or antibioticswhich can be applied, e.g., sprayed, onto transgenic plants. Inducibleexpression of the amylase-producing nucleic acids of the invention willallow the grower to select plants with the optimal starch/sugar ratio.The development of plant parts can thus controlled. In this way theinvention provides the means to facilitate the harvesting of plants andplant parts. For example, in various embodiments, the maize In2-2promoter, activated by benzenesulfonamide herbicide safeners, is used(De Veylder (1997) Plant Cell Physiol. 38:568-577); application ofdifferent herbicide safeners induces distinct gene expression patterns,including expression in the root, hydathodes, and the shoot apicalmeristem. Coding sequences of the invention are also under the controlof a tetracycline-inducible promoter, e.g., as described with transgenictobacco plants containing the Avena sativa L. (oat) argininedecarboxylase gene (Masgrau (1997) Plant J. 11:465-473); or, a salicylicacid-responsive element (Stange (1997) Plant J. 11:1315-1324).

If proper polypeptide expression is desired, a polyadenylation region atthe 3′-end of the coding region should be included. The polyadenylationregion can be derived from the natural gene, from a variety of otherplant genes, or from genes in the Agrobacterial T-DNA.

Expression Vectors and Cloning Vehicles

The invention provides expression vectors and cloning vehiclescomprising nucleic acids of the invention, e.g., sequences encoding theamylases of the invention. Expression vectors and cloning vehicles ofthe invention can comprise viral particles, baculovirus, phage,plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes,viral DNA (e.g., vaccinia, adenovirus, foul pox virus, pseudorabies andderivatives of SV40), P1-based artificial chromosomes, yeast plasmids,yeast artificial chromosomes, and any other vectors specific forspecific hosts of interest (such as bacillus, Aspergillus and yeast).Vectors of the invention can include chromosomal, non-chromosomal andsynthetic DNA sequences. Large numbers of suitable vectors are known tothose of skill in the art, and are commercially available. Exemplaryvectors are include: bacterial: pQE vectors (Qiagen), pBluescriptplasmids, pNH vectors, (lambda-ZAP vectors (Stratagene); ptrc99a,pKK223-3, pDR540, pRIT2T (Pharmacia); Eukaryotic: pXT1, pSG5(Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia). However, anyother plasmid or other vector may be used so long as they are replicableand viable in the host. Low copy number or high copy number vectors maybe employed with the present invention.

The expression vector can comprise a promoter, a ribosome binding sitefor translation initiation and a transcription terminator. The vectormay also include appropriate sequences for amplifying expression.Mammalian expression vectors can comprise an origin of replication, anynecessary ribosome binding sites, a polyadenylation site, splice donorand acceptor sites, transcriptional termination sequences, and 5′flanking non-transcribed sequences. In some aspects, DNA sequencesderived from the SV40 splice and polyadenylation sites may be used toprovide the required non-transcribed genetic elements.

In one aspect, the expression vectors contain one or more selectablemarker genes to permit selection of host cells containing the vector.Such selectable markers include genes encoding dihydrofolate reductaseor genes conferring neomycin resistance for eukaryotic cell culture,genes conferring tetracycline or ampicillin resistance in E. coli, andthe S. cerevisiae TRP1 gene. Promoter regions can be selected from anydesired gene using chloramphenicol transferase (CAT) vectors or othervectors with selectable markers.

Vectors for expressing the polypeptide or fragment thereof in eukaryoticcells can also contain enhancers to increase expression levels.Enhancers are cis-acting elements of DNA, usually from about 10 to about300 bp in length that act on a promoter to increase its transcription.Examples include the SV40 enhancer on the late side of the replicationorigin by 100 to 270, the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, and theadenovirus enhancers.

A nucleic acid sequence can be inserted into a vector by a variety ofprocedures. In general, the sequence is ligated to the desired positionin the vector following digestion of the insert and the vector withappropriate restriction endonucleases. Alternatively, blunt ends in boththe insert and the vector may be ligated. A variety of cloningtechniques are known in the art, e.g., as described in Ausubel andSambrook. Such procedures and others are deemed to be within the scopeof those skilled in the art.

The vector can be in the form of a plasmid, a viral particle, or aphage. Other vectors include chromosomal, non-chromosomal and syntheticDNA sequences, derivatives of SV40; bacterial plasmids, phage DNA,baculovirus, yeast plasmids, vectors derived from combinations ofplasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl poxvirus, and pseudorabies. A variety of cloning and expression vectors foruse with prokaryotic and eukaryotic hosts are described by, e.g.,Sambrook.

Particular bacterial vectors which can be used include the commerciallyavailable plasmids comprising genetic elements of the well known cloningvector pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala,Sweden), GEM1 (Promega Biotec, Madison, Wis., USA) pQE70, pQE60, pQE-9(Qiagen), pD10, psiX174 pBluescript II KS, pNH18A, pNH16a, pNH18A,pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233-3, DR540, pRIT5(Pharmacia), pKK232-8 and pCM7. Particular eukaryotic vectors includepSV2CAT, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL(Pharmacia). However, any other vector may be used as long as it isreplicable and viable in the host cell.

The nucleic acids of the invention can be expressed in expressioncassettes, vectors or viruses and transiently or stably expressed inplant cells and seeds. One exemplary transient expression system usesepisomal expression systems, e.g., cauliflower mosaic virus (CaMV) viralRNA generated in the nucleus by transcription of an episomalmini-chromosome containing supercoiled DNA, see, e.g., Covey (1990)Proc. Natl. Acad. Sci, USA 87:1633-1637. Alternatively, codingsequences, i.e., all or sub-fragments of sequences of the invention canbe inserted into a plant host cell genome becoming an integral part ofthe host chromosomal DNA. Sense or antisense transcripts can beexpressed in this manner. A vector comprising the sequences (e.g.,promoters or coding regions) from nucleic acids of the invention cancomprise a marker gene that confers a selectable phenotype on a plantcell or a seed. For example, the marker may encode biocide resistance,particularly antibiotic resistance, such as resistance to kanamycin,G418, bleomycin, hygromycin, or herbicide resistance, such as resistanceto chlorosulfuron or Basta.

Expression vectors capable of expressing nucleic acids and proteins inplants are well known in the art, and can include, e.g., vectors fromAgrobacterium spp., potato virus X (see, e.g., Angell (1997) EMBO J.16:3675-3684), tobacco mosaic virus (see, e.g., Casper (1996) Gene173:69-73), tomato bushy stunt virus (see, e.g., Hillman (1989) Virology169:42-50), tobacco etch virus (see, e.g., Dolja (1997) Virology234:243-252), bean golden mosaic virus (see, e.g., Morinaga (1993)Microbiol Immunol. 37:471-476), cauliflower mosaic virus (see, e.g.,Cecchini (1997) Mol. Plant Microbe Interact. 10:1094-1101), maize Ac/Dstransposable element (see, e.g., Rubin (1997) Mol. Cell. Biol.17:6294-6302; Kunze (1996) Curr. Top. Microbiol. Immunol, 204:161-194),and the maize suppressor-mutator (Spm) transposable element (see, e.g.,Schlappi (1996) Plant Mol. Biol. 32:717-725); and derivatives thereof.

In one aspect, the expression vector can have two replication systems toallow it to be maintained in two organisms, for example in mammalian orinsect cells for expression and in a prokaryotic host for cloning andamplification. Furthermore, for integrating expression vectors, theexpression vector can contain at least one sequence homologous to thehost cell genome. It can contain two homologous sequences which flankthe expression construct. The integrating vector can be directed to aspecific locus in the host cell by selecting the appropriate homologoussequence for inclusion in the vector. Constructs for integrating vectorsare well known in the art.

Expression vectors of the invention may also include a selectable markergene to allow for the selection of bacterial strains that have beentransformed, e.g., genes which render the bacteria resistant to drugssuch as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycinand tetracycline. Selectable markers can also include biosyntheticgenes, such as those in the histidine, tryptophan and leucinebiosynthetic pathways.

Host Cells and Transformed Cells

The invention also provides a transformed cell comprising a nucleic acidsequence of the invention, e.g., a sequence encoding an amylase of theinvention, or a vector of the invention. The host cell may be any of thehost cells familiar to those skilled in the art, including prokaryoticcells, eukaryotic cells, such as bacterial cells, fungal cells, yeastcells, mammalian cells, insect cells, or plant cells. Exemplarybacterial cells include E. coli, Streptomyces, Bacillus subtilis,Salmonella typhimurium and various species within the generaPseudomonas, Streptomyces, and Staphylococcus. Exemplary insect cellsinclude Drosophila S2 and Spodoptera Sf9. Exemplary animal cells includeCHO, COS or Bowes melanoma or any mouse or human cell line. Theselection of an appropriate host is within the abilities of thoseskilled in the art. Techniques for transforming a wide variety of higherplant species are well known and described in the technical andscientific literature. See, e.g., Weising (1988) Ann. Rev. Genet.22:421-477, U.S. Pat. No. 5,750,870.

The vector can be introduced into the host cells using any of a varietyof techniques, including transformation, transfection, transduction,viral infection, gene guns, or Ti-mediated gene transfer. Particularmethods include calcium phosphate transfection, DEAE-Dextran mediatedtransfection, lipofection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

In one aspect, the nucleic acids or vectors of the invention areintroduced into the cells for screening, thus, the nucleic acids enterthe cells in a manner suitable for subsequent expression of the nucleicacid. The method of introduction is largely dictated by the targetedcell type. Exemplary methods include CaPO₄ precipitation, liposomefusion, lipofection (e.g., LIPOFECTIN™), electroporation, viralinfection, etc. The candidate nucleic acids may stably integrate intothe genome of the host cell (for example, with retroviral introduction)or may exist either transiently or stably in the cytoplasm (i.e. throughthe use of traditional plasmids, utilizing standard regulatorysequences, selection markers, etc.). As many pharmaceutically importantscreens require human or model mammalian cell targets, retroviralvectors capable of transfecting such targets are preferred.

Where appropriate, the engineered host cells can be cultured inconventional nutrient media modified as appropriate for activatingpromoters, selecting transformants or amplifying the genes of theinvention. Following transformation of a suitable host strain and growthof the host strain to an appropriate cell density, the selected promotermay be induced by appropriate means (e.g., temperature shift or chemicalinduction) and the cells may be cultured for an additional period toallow them to produce the desired polypeptide or fragment thereof.

Cells can be harvested by centrifugation, disrupted by physical orchemical means, and the resulting crude extract is retained for furtherpurification. Microbial cells employed for expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents. Suchmethods are well known to those skilled in the art. The expressedpolypeptide or fragment thereof can be recovered and purified fromrecombinant cell cultures by methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Protein refolding steps can be used, asnecessary, in completing configuration of the polypeptide. If desired,high performance liquid chromatography (HPLC) can be employed for finalpurification steps.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts and other cell linescapable of expressing proteins from a compatible vector, such as theC127, 3T3, CHO, HeLa and BHK cell lines.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence. Dependingupon the host employed in a recombinant production procedure, thepolypeptides produced by host cells containing the vector may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay or may not also include an initial methionine amino acid residue.

Cell-free translation systems can also be employed to produce apolypeptide of the invention. Cell-free translation systems can usemRNAs transcribed from a DNA construct comprising a promoter operablylinked to a nucleic acid encoding the polypeptide or fragment thereof.In some aspects, the DNA construct may be linearized prior to conductingan in vitro transcription reaction. The transcribed mRNA is thenincubated with an appropriate cell-free translation extract, such as arabbit reticulocyte extract, to produce the desired polypeptide orfragment thereof.

The expression vectors can contain one or more selectable marker genesto provide a phenotypic trait for selection of transformed host cellssuch as dihydrofolate reductase or neomycin resistance for eukaryoticcell culture, or such as tetracycline or ampicillin resistance in E.coli.

Amplification of Nucleic Acids

In practicing the invention, nucleic acids of the invention and nucleicacids encoding the polypeptides of the invention, or modified nucleicacids of the invention, can be reproduced by amplification.Amplification can also be used to clone or modify the nucleic acids ofthe invention. Thus, the invention provides amplification primersequence pairs for amplifying nucleic acids of the invention. Inalternative aspects, where the primer pairs are capable of amplifyingnucleic acid sequences including the exemplary SEQ ID NO:1, or asubsequence thereof; a sequence as set forth in SEQ ID NO:3, or asubsequence thereof; a sequence as set forth in SEQ ID NO:5, or asubsequence thereof; a sequence as set forth in SEQ ID NO:7, or asubsequence thereof, a sequence as set forth in SEQ ID NO:9, or asubsequence thereof, a sequence as set forth in SEQ ID NO:11, or asubsequence thereof, a sequence as set forth in SEQ ID NO:13, or asubsequence thereof, a sequence as set forth in SEQ ID NO:15, or asubsequence thereof. One of skill in the art can design amplificationprimer sequence pairs for any part of or the full length of thesesequences; for example:

The exemplary SEQ ID NO:1 is

atgagagttt cctccatagg aaatggcaga atgctgataa actttgatga gaaaggaaga 60atagtcgata tttattatcc ttatatagga atggagaacc agacttctgg aaacccaatt 120aggttagcta tttgggacaa agataagaaa gtggcatctc tagatgagga ttgggaaact 180actgtattat atatagatga agctaatatg gttgagatta ggagtgatgt taaggagtta 240ggactttctc ttctctctta taactttcta gattctgatg atccgatata tatgtctatt 300gtaaaaatag caaataacga aaataatagc agaaatataa aagtattttt tatacatgat 360ataaatttat attcaaaccc ttttggggac actgcattct atgatcccct acccctttca 420attatacatt ataagtctaa acgatattta gcctttaaag tgtttaccac ggtatcgaca 480ctttctgagt ataacatagg caaaggtgac ttaattggag atatttatga tggcaattta 540ggacttaatg gtatagaaaa tggtgatgta aattcaagta tgggtataga gataaatata 600gatcctaatt cctatttgaa attatactac gtaatagtcg cagatagaaa cttggaaggc 660ttaaggcaaa aaataaggaa aataaacttt gcaaacgtag agacatcgtt tacgttaacc 720tatatgtttt ggcggaattg gttaaagaaa aataaactct tcagaaataa tttaatgcag 780gatattaaga gagtctatga tgtgagtctt tttgtgataa gaaatcacat ggacgttaac 840gggtcaataa tagcttcctc agacttctcc ttcgtcaaga tttatgggga ctcatatcag 900tattgttggc ctagagatgc ggcaattgca gcttatgctc tagatctagc tggctataag 960gaactagcat taaaacactt ccagttcatt tctaatattg caaattctga aggcttccta 1020tatcataaat ataatccaaa tacaactcta gctagttctt ggcatccttg gtattataaa 1080ggtaaaagga tatacccaat tcaaggggat gagacggcat tagaagtatg ggcaatagct 1140agtcattacg aaaaatatga agatattgac gaaatacttc cattatataa gaagttcgtg 1200aagccagcct taaaatttat gatgtctttt atggaagaag gattgccaaa accttctttt 1260gacctatggg aagaaaggta tggtatacat atttacacag tatctacggt ttacggcgca 1320ttaacaaagg gagcaaagtt agcttatgat gtaggtgatg aaatattaag tgaagattta 1380agtgatacat cgggtttatt aaaaggaatg gttttgaaaa gaatgactta taatggaaga 1440tttgttagaa gaatagacga ggaaaataac caagatctaa ctgtggactc aagtctctat 1500gctccattct tctttggtct tgttaatgca aatgacaaaa tcatgataaa taccattaac 1560gagattgaaa gcagattaac tgtgaatggc gggataataa ggtatgagaa tgatatgtat 1620cagaggagga aaaaacaacc aaacccttgg ataattacga cattatggct atctgaatat 1680tatgcaacaa ttaacgataa aaataaggca aacgagtaca taaaatgggt aattaatagg 1740gcattaccaa ccggcttttt accagaacaa gttgatccag aaacttttga gccaacttca 1800gttacacctt tggtatggtc tcatgctgaa ttcataatag caattaataa catt 1854

Thus, an exemplary amplification primer sequence pair is residues 1 to21 of SEQ ID NO:1 (i.e., atgagagtttcctccatagga) and the complementarystrand of the last 21 residues of SEQ ID NO:1 (i.e., the complementarystrand of ataatagcaattaataacatt).

The exemplary SEQ ID NO:3 is

atggttaggt atacaccgct tggcaatggg cggttgctta tagcttttga tactgattac   60aggattgttg atttttacta ttcaaagttt gcctccgaaa atcattcgtc tggtcatcca  120ttctactttg gtgtttccgt ggatggcaat ttcaactgga tagacagaaa tgcaatcaag  180cacatggact actacgacca caccatggtc tctgtcgtca actacacgca taacggtatt  240gatttcgaga acagggatat ggttgacata tacaaggaca tctttattag gcgggtggtt  300gctgaaaaca agaccggtaa ggatgtaaac ctgaagatct tctttcacca gaatttctac  360atatatggca atgacatagg ggataccgct gcttactttc ctgaataccg cggtgtgatc  420cattataagg gagggagata ctttctcgca tccactcttg atgagagcgg taatttctgc  480gatcaatatg ccacaggggt taaggatgtg ggtgagctga agggcacatg gaaggatgcc  540gaggacaatg aattatcaat gaacccggtg gcaataggtt cggtggattc tgtcataagg  600cattccacga ctctgaaggc cggttcaaag ttcacgctct attatttcat catagcggga  660agaaacatca acgatataga gagcgaatat tcaaatgtga atgtccagta cctccaaaag  720cttctgagga gaacaacaaa ctactgggag ctctggtctt cgaaggtgac tcccagcctg  780gattcagaca caacagcgct ttaccgcaga tcgctcttcg tgactaagag ccacgcaaac  840gatcttgggg ccatagccgc atcctgcgac agcgatatac tgaagctgag ccatgacgga  900tactactacg tctggcccag ggatgcctcc atggctgcat acgccttgag catatccggg  960cacagcgaaa ccgccagacg cttctttgcc ctgatggaag attcactttc agaagaggga 1020tacctgtacc acaaatacaa cgtcgacggc aagatcgcca gcagctggtt accgcacgtc 1080atgaatggca aatccatata tccaatacag gaggatgaaa cagctctggt ggtctgggca 1140ctctgggaat actttaggaa gtacaatgat atcggcttca ccgcaccgta ttatgaacgc 1200cttataacca gggcagcaga ctttatgacc aattttgttg acaacaacgg ccttcccaag 1260ccatcctttg atctgtggga agagcgctat ggaatccatg cctacactgt tgctacggtt 1320tatgccgccc tgaaagcagc ttcaaacttt gcaaacgttt tcggcgatcc tgatctatcg 1380gaaaaatacg aaaatgctgc ggaaaggatg taccatgcgt tcgatgaaag gttctattct 1440gaggatacgg gatactatgc aagggccatc atagacggaa agccggactt caccgtggac 1500agcgccctca cctcactggt gctctttgga atgaaggatg cggacgatcc aaaggttatt 1560tctaccatgc agaggatatc tgaagaccta tgggtgaatg gcgttggagg catagcgcgc 1620taccagaacg acagatacat gagggtgaag gacgatccaa gcgttcctgg aaatccctgg 1680ataataacca cgctgtggat ggcaagatac tatatgcgtt ttggtgattt tgaaaaggcc1740 1572

Thus, an exemplary amplification primer sequence pair is residues 1 to21 of SEQ ID NO:3 (i.e., atggttaggt atacaccgctt) and the complementarystrand of the last 21 residues of SEQ ID NO:3 (i.e., the complementarystrand of tttggtgattttgaaaaggcc).

The exemplary SEQ ID NO:5 is

atgatttata tgggcggaat aattggaaat aacaacctat tagtaaaaat cggagattat 60ggggaaatta gttatgtttt ctatcctcat gtgggttatg aaacccattt cttcgattct 120gcattggcag tgtatgataa aaaagtaaaa tggcattggg atgatgattg ggacatctct 180caaaaatata ttgaagaaac aaatatattc aaaactatac tggaagacga taaaataata 240ttgacaatta aagattttgt tccagtttcg cacaatgtaa tcattagaag gttgcatata 300aaaaataaac tcgataaaaa attgaatttt aagctatttt tttatgaaaa tttaaggatt 360ggggagtatc ctacagaaaa tgccgtaaga tttttagagg atgagggatg tatcgttaaa 420tataacgaaa aatatgtttt ctgcattgga agtaataaaa agatagattc gttccagtgt 480ggaaacagat acagcaaaaa cagtgcatac gtagatattg aaaacggatt gttgatggaa 540cataaagaaa gccatggact gatgacagat agtgcaatat cgtggaatat agagattgat 600aaaggaaaga gcttagcgtt taatatctat atacttctac aaaaatttga tggagattta 660tcaataataa ccgagcagtt aaagattata atgaacaata ctgtacatat caaagacctt 720tcaatgaact attggaaaaa tagcattgga aatataaaag aacatatcca tcctcaattt 780cattcagata aagaaatatg tcctatagct aaaagggctt taatggttct tctaatgctt 840tgtgataaag atggggggat tatagccgct ccttcactac atccagacta taggtatgtt 900tgggggaggg atggggctta tatagcaatt gcattagatt tatttggaat tagaggaatt 960cccgatagat tctttgaatt catgtctaaa attcaaaatg atgatggttc atggctacaa 1020aactactaca caaatggaaa accgagatta acagcgatgc agattgacca aattggctct 1080atactgtggg ctatggatgt gcattataga ttaactggaa atagaaagtt tgttgagagg 1140tattggaata ctatagaaaa agctggaaat tatctaactt ctgccgcttt aaacttcaca 1200ccatgctttg atttatggga agaaaagttt ggagtttttg catatactat gggagcaatc 1260tatgcgggat taaaagctgc ttatagtatg agtaaagctg ttgatatgag ggataaggtt 1320aaacattggg aaaaagctat tgaatttttg aaaaaggaag ttccaaggag attttattta 1380gaagatgagg aaagatttgc taaatcaata aatccattgg ataaggagat agacgctagc 1440atattgggat tgagctatcc atttaactta attgatgttg atgatgaaag gatgataaaa 1500acagctgagg ctattgaaaa tgcatttaac tacaaagttg gtgggattgg gagatatcct 1560aatgatgttt attttggagg gaacccatgg attataacga cattgtggat ttctttatat 1620tatagaaggt tatccaaggt tttaaaagag aaaaataaaa atgatatggc agagaaatat 1680ttaaaaaaat ctaaaaaatt gtttgattgg gcagttaaat acagctttaa cggtttgttt 1740ccagagcaga tacataagga cctcggcatt ccaatgtctg caatgccttt gggctggagc 1800aatgcaatgt ttttaatcta cctatataag gatgacaatg tcataattcc ataa 1854

Thus, an exemplary amplification primer sequence pair is residues 1 to21 of SEQ ID NO:5 (i.e., atgatttatatgggcggaata) and the complementarystrand of the last 21 residues of SEQ ID NO:5 (i.e., the complementarystrand of gacaatgtcataattccataa).

The exemplary SEQ ID NO:7 is

atggcaggga ttattggaaa tggaaaccta ctggcaaaaa ttgatgactt ggggtctata 60gaatatatat ttttcccaca tttgggttac gaaacacata ttctcgatac atcatttgct 120atatactaca acaacaaaat aaaatggcat tgggatcata gttgggacgt tagtcagaac 180tatctcaaag attccaacat attaaaaaca acttatgaaa atgatgactt cttaatatat 240tctaaggatt gtgtatccat atctcacaac cttattgtta aacaactttc tataataaac 300aagaccaatt cagaaaagga cataaaatta tttttttatg aaaatttgag aataggtgaa 360acgccgagta aaagcactgt aaaatttgtt aaagaaaaaa actgcctaat taaacatgac 420aaaaattata ttttctgtat tggcagtaat aaaaaagtat cctcttacca atgtgggatt 480aaatactctg agagtagtgc tttaagggac attgaaaatg gagtactgaa agagcagagt 540tccgccacag gattaatcac agacagtgcc ctttgctggg aattcaaaat caaacctaac 600caaaaataca ctctttcaat actcatactt cctgaaaagt atgatggtga ttataataaa 660accctaaact taatggatac tctacacatg gtaaaagaca acctcaaaga cctatataac 720\ctcacaagaa atttctggaa aagtagagta gatagcatgg taaataagtg gggaatctta 780aagttggaag aatataaaga atgcatagat atatgcaaaa gatctctact aaccctatta 840cttctctgcg attataaggg gggaataatt gcttctcctt ctttacatcc agattatagg 900tatgtctggt gtagggatgc agggtatatg gcagttgcgt tggatttgtg tgggcagcat 960gaaatgagtg agaaatactt tgagtggtgc aagacaacac aaaacagtga cggttcttgg 1020gttcaaaatt actatgtgga ggggtatcca agattcacag ccatccaaat agatcaggtg 1080ggtactacca tttgggcact tcttgtgcac tatagaataa ctggagacaa acatttttta 1140aaaagaaatt gggaaatggt caaaaaagca ggggactatt tgagcagagc tgctgaccaa 1200ttaataccct gctatgactt atgggaagaa aagtttgggg tctttgcata taccctcgga 1260gcaatatatg gggggttgaa atcaggttat ttaattggaa aagaacttga caaagaagaa 1320gaaatacage attggaaaaa aagcatgaac ttccttaaaa atgaagtggt aaatagactc 1380tacttaaaaa atgagaagag gtttgcaaaa tcattaaaac cattagataa aaccatagat 1440acgagtattt tagggttaag tttcccctat ggacttgtgt cagtcgatga cccaagaata 1500atatcaactg caaatcagat tgaaaaagcc ttcaactaca aagttggtgg tgttggtaga 1560tatccagagg acatatactt tggaggaaat ccttggataa taacaaccct atggctctat 1620atgtattata aaaagttagt tgatacatta tcaaaaaaag gaaaattcca agagtccata 1680attgataatt acaataaaaa atgttacaac ttgcttaaat ggattctaaa acatcaattc 1740aatggtatgt ttccagaaca agtccataaa gatttgggaa ttccaatatc tgcaattccc 1800cttggctggt cacatgccat ggttataatc gctattcatg gtgattacga catcctaata 1860ccctaa 1866

Thus, an exemplary amplification primer sequence pair is residues 1 to21 of SEQ ID NO:7 (i.e., atggcagggattattggaaat) and the complementarystrand of the last 21 residues of SEQ ID NO:7 (i.e., the complementarystrand of a tacgacatcctaataccctaa).

The exemplary SEQ ID NO:9 is

atgatttata tgggtggaat cgttggaaac aatagtttat tagccaaaat tggagattat 60ggggaaattg aatacctttt ttatccccaa gttggttatg aaactcattt ctttgactct 120gcattggcag tttatgataa aaaagtaaag tggcattggg atgatgattg ggatataacc 180caaaaataca ttgaggaaac gaacatattt aaaactatct tagaagatga taagattata 240ttaaccatta aagattttgt gccagtatct cacaacgtgc ttataagaag agtgtatata 300aaaaataaac tcgataaaaa attaaatttt aagctctttt tttacgaaaa tttgagaatt 360ggtgaaaacc caataacaaa tacagttaaa ttcttagaag atggttgtat cgttaaatat 420aatggaaaat atattttttg cattggaagt gataaaagaa tagattcatt tcagtgtgga 480aatagataca gtaaaacaag tgcttacata gacatagaaa atgggatatt gaaggagcat 540aaagagagtt ctggattatt aaccgatagt gcaatatcat ggaatataaa gattgatgaa 600aaaagaagtt tggcattcaa catctacata cttccacaaa gattcgatgg agatttttca 660ataataactg aacaactaaa gattataatg aataacagtg aaaacattaa aaatctctca 720atgaattatt ggaaacatat tataggggag ataaatagat ttatacatcc tgagcttagg 780caaaataata agatttattc tataactaaa agggctttaa tgacactttt aatgttatgt 840gataaggaag gagggattat agcggctcca tctctacatc cagattatag atacgtgtgg 900ggaagagatg gaagttatat ctcaattgct ttggacttat ttggcataag gaacattcca 960gacagatttt ttgaattcat gtctaagata caaaatgcag acggttcatg gctacaaaat 1020tattatgtta atggaaaacc acgattaact gcaatacaga ctgaccaaat tggttccata 1080ttatgggcaa tggatgtgca ttacagatta actggggata gaaagttcgt tgagagatac 1140tggaacacta tagagaaagc tgctaattat ttaaggttgg tagctttaaa ctttactcca 1200tgcttcgatt tgtgggaaga gaggtttgga gtatttgctt atacaatggg agctacttac 1260gctggattga aatgtgcata cagcatgagt aaggcagtga ataaaaggga taaagttaag 1320gattggggaa aaaccataga atttttaaaa catgaggttc caaagagatt ttatttggaa 1380gatgaggaaa gatttgctaa atcaataaat cctttagaca agacgataga cacaagcata 1440ttgggtttaa gttacccttt caatttgatt gatgttgatg atgagagaat gataaaaaca 1500gccgaagcaa ttgaaaaagc tttcaaatat aaggttggag ggattgggag atatccagaa 1560gacatttact ttggaggcaa tccatggatt ataaccacat tatggctttc tttgtattat 1620agaaggttat acaaggtttt aaaagaaaaa gatgataatg gggcagatat ttatctacaa 1680aaatctaaga agttgtttaa ttgggtgatg aaatacagct ttgatgggct gtttccagag 1740caaattcata aagaattagg tgtgccaatg tccgctatgc ctttaggctg gagcaatgca 1800atgttcctca tttatgtgta tgagaatgat aaggtcataa taccataa 1848

Thus, an exemplary amplification primer sequence pair is residues 1 to21 of SEQ ID NO:9 (i.e., atgatttatatgggtggaatc) and the complementarystrand of the last 21 residues of SEQ ID NO:9 (i.e., the complementarystrand of gataaggtcataataccataa).

The exemplary SEQ ID NO:11 is

gtggttagca tggtaggaat tattgggaat gggaaaatcc tcgcaaagat tgatgactca 60ggctctttgg aatacatatt ttttccacat ttggggcatg agaaacatat ttttgattca 120tcatttgcca tattttatga taataagttg aaatggaatt gggacaattc ctgggatatt 180aatcagaact atttaaaaga tacaaacata ttgaaaacat catatgaaaa cgaggatttt 240ctaatagaat caaaggacta cgtgcctata tcccataact cgataattaa gcaaatatca 300atattaaaca aatccagcga aaaaaagaat ttaaaactgt ttttttatga aaatttaaga 360atgggagaaa ttcctgaagt aagtactgta aagtatagaa agaacaggga ggggattatt 420aaatacgata agaattatgt tttttgtatc ggcagtaata aaaaagtatc ttcataccaa 480tgtggtgtta ggtcatccga gagtagtgcc ctaaatgatc tcaaaaatgg tattttaaag 540gaatacgata gtgctgaagg cctaatcaca gatagcgcac tgggttggga ccttgagttg 600agtccaaatc aggaacagaa agtctcaata tttatatttg cagataagta tggtggggat 660tataccaaaa ttatgaattt attggataca ctaaatatag ttataaccaa tcacgcagac 720atatatgatc ttacaatggc atactggaag aacatgattg aaaccactgc gaatagtcta 780tgcaattcaa atcaagtctt taaagattta acacatataa aagacgacgc aaatatttca 840aatttaaaaa gaataaaaca gtatgaagct atttgtaaaa gatccctatt aaccatttta 900ctcctttgtg atcataatgg tggaataatt gcatcaccat cactctatcc agattataga 960tatgtatggt gtagggacgc aggttatatg gccgtcgcac ttgacctatg tggtcagcat 1020ggaataagcg aaaaatactt tgaatggtgc aaaaaaacac aaaatagtga tggctcatgg 1080gttcaaaact actacgtaga aggaaatcca aggcttacgg caattcaaat tgaccaagtt 1140ggtactacaa tctgggccgc acttgtacat tatagaataa ctagggacaa attatttctg 1200aacagatatt gggaaatgat taaaaaagca ggggattatt taagtagtgt tgccaatcca 1260ccatcaccaa gctatgattt atgggaagaa aaatatggtg tattcgcata cacacttggc 1320gcaatttatg gaggattaaa atctgcctac aacatttgta aaatactggg caaggaagaa 1380cacgatatcc aaaattggaa agagagcatg gacttcctta aaaacgaaat ggtagatagg 1440ctttatttaa aagatgaaaa tagatttgca aaatcattgg atccattgga caaagctcta 1500gatgctagta ttttagggct cagttttcca tataatttgg tacctgttga tgaccctaga 1560atgattagca ccgccaacca aattgaaaat gcgtttaagt ataaggttgg aggtatagga 1620aggtaccctg aagatgttta tttcggaggg aatccttgga taataaccac aatatggctc 1680catatgtact atgaaaactt gattaaatca ttatctaaac atggtaaaaa tgccatacat 1740tctgatcaaa tccctgattc ttcaggggac cttaaggatt ttgtctcaat tatagggtcc 1800attgaaaacc atggtgaaaa gtcagatgaa acccctagtt ccgacacact ccttacttat 1860gcccaaaaat gtaacaattt gtttgattgg actttaaagt ataactttaa tgaactattt 1920ccagaacagg ttcacaaaga tcttggagct ccgatatctg caattccact tgggtggtca 1980catgcaatgg tcataattgc catccatggt aactttgata tattaatacc ttaa 2034

Thus, an exemplary amplification primer sequence pair is residues 1 to21 of SEQ ID NO:11 (i.e., gtggttagcatggtaggaatt) and the complementarystrand of the last 21 residues of SEQ ID NO:11 (i.e., the complementarystrand of tttgatatattaataccttaa).

The exemplary SEQ ID NO:13 is

atgattgttg gtaataatag ctttttatgt aagatagggg atcatggaga aattgaatat 60gcattctacc cccatgttgg ttatgaacta catttttttg atagttcttt agctatatat 120gataaagaaa ttatgtggat atgggataaa gagtggagtg tatatcagaa atatattgag 180gacactaata tattcaaaac tactttagaa aatgagaata tcatatttgt tataaaagat 240ttagtcccaa tttcacataa tgtattaatt aggagagttt tcattaaaaa taaacttcca 300tataattata attttaaact atttttctat gaaaatctta gaattggaga acatccttca 360gaaaatacag ttaagttttt agatgattgt atagttaaat ttaatggcaa atatactttt 420tgtataagca gtgataaaaa aataaattca tatcagtgtg gaaatagata tagtgaaaaa 480tctgcttata aagatattga aaatggttta ttatctgaaa atcctgaaag tgttggagtt 540ctaactgaca gtgctattga atgggatata gatttaaaac cacatggaaa agtagcattt 600aacatctaca tctttcctca tattggaaat aatatagaga ttataaaaaa tcagttaaat 660attattaaaa atctctcttc tgaaataaaa aatatatctc taaattattg gaagagttct 720tttgatataa aaggttatct atttaatgaa aaatatttaa aattagcaaa aagggcttta 780atgatactaa caatgctttc tgacaaaaat ggaggaatta tagcctctcc atctattcat 840cctgattata gatatgtttg gggtagagat ggaagttata tggctgtggc attatccatt 900tatggaataa aaaacattcc atggaggttc ttccatttca tgtctaaagt ccagaatctt 960gatggttcat ggttacaaaa ctattataca gatggtaaac caagattaac tgctttacaa 1020atagatcaaa taggttcagt tctttgggct atggaagttt attatagaac tacaggtgat 1080agagagtttg ttaaaaaatt ctgggaaact attgagaaag ctggaaattt cttatataat 1140gcttcattat ctttaatgcc atgttttgat ctttgggaag aaaaatatgg ggtattttca 1200tatactttag gagcaatgta tggaggatta agggcaggat gtagtttagc taaagctata 1260gaagagaaaa aagaagattg gaaaaaggct ttagataaat taaagaagga tgttgattta 1320ttatatttaa gtgatgaaga aagatttgtt aaatctatta acccattgaa caaagagatt 1380gatacaagta tattagggct tagctatcca tttggactag ttaaagttaa tgatgaaaga 1440atgataaaaa ctgctgaagc catagaaaaa gcttttaaat acaaagttgg aggtattggg 1500agatatccat ctgatgttta ttttggagga aatccttgga ttataacaac actttggtta 1560gctttatatt atagaagact atttattact acaaatgata gaaaatattt agaaaaatca 1620aaaaagctat ttaattgggt tattaaccat atctatctat tccctgaaca gatacataaa 1680gaattagcta ttcctgtatc agctatgcct ttaggttgga gttgtgctat gctgttattc 1740tatctatata aaaatgatga cataatagtg ataaaatga 1779

Thus, an exemplary amplification primer sequence pair is residues 1 to21 of SEQ ID NO:13 (i.e., atgattgttggtaataatagc) and the complementarystrand of the last 21 residues of SEQ ID NO:13 (i.e., the complementarystrand of gacataatagtgataaaatga).

The exemplary SEQ ID NO:15 is

atgaaattga atagaaaact tataaaatat ttacccgtac tatttcttgc gtccagtgtg 60ctaagtggat gcgctaacaa taatatatca aacattaaaa ttgagagatt gaataatgta 120caagcagtaa atggccctgg agaggctgat acttgggcta aagctcagaa acaaggtgta 180gggactgcaa acaactatac ttccaaagta tggtttacca ttgcagacgg ggggatatct 240gaggtttact atccgactat agatactgct gatgtaaagg atattaaatt ttttgtgaca 300gatggaaaaa cgtttgtctc agatgagaca aaagacacaa taaccaaagt cgaaaagttt 360actgaaaaat cgttggggta taaaatcatt aacacagata aagaagggag atataagata 420actaaagaaa tatttacgga tgtaaagagg aattctctcg taattaaaac gaagtttgaa 480gccttaaaag gcaatgttga tgattacagg ctttacgtaa tgtgtgatcc tcatgtaaaa 540aatcagggca aatataatga aggatatgca gttaaggcaa atggcaatgt tgcgctaatt 600gctgaaagag atggaattta cactgcattg tcatctgaca taggatggaa aaagtattcg 660atagggtatt ataaagtaaa tgacattgag accgatcttt ataaaaatat gcaaatgact 720tacaattacg acagtgcaag aggcaacatc atagaaggtg ctgagataga tcttaagaaa 780aacaggcaat ttgaaatcgt tctgtctttc ggacagagtg aagacgaggc agtaaaaaca 840aacatggaaa ctttaaatga taattatgac agcttaaaga aagcgtatat agaccaatgg 900gagaagtatt gcgatagcct taatgacttt ggaggaaaag caaattcact gtattttaac 960agtatgatga tattaaaggc cagtgaagac aagacaaaca aaggtgctta tatagcatcg 1020ctatctattc cgtggggtga tggccaagaa gatgacaata ttggtggcta ccatctcgta 1080tggtcaagag atctgtacca tgtagcgaat gcatttattg ttgctggtga tactgattcg 1140gcaaatagag cactggatta tttagacaaa gtagtgaaag acaatggaat gattcctcaa 1200aatacatgga taaatggaag gccttattgg acaggcatac agcttgatga gcaggcggat 1260ccaataatat taagctatag gttgaaaaga tacgatctct atgaaagtct tgttaagcct 1320ttggcggatt tcatcatgaa aataggccct aagacgggac aagaaagatg ggaagaaata 1380ggtggatatt cgccagcaac attggcttca gaagtagctg gacttacatg tgctgcgtat 1440atagctgaac aaaataagga ctttgaatct gctaaaaaat atcaagaaaa ggcggataat 1500tggcaaaggc ttattgacaa cctaacttac acagaaaaag gcccattggg agatggtcac 1560tattatataa ggatagcagg gcttccagat ccaaatgccg atttcatgat aagcatagcg 1620aatggcggtg gtgtatacga ccaaaaagaa atcgtggatc caagttttct ggaacttgta 1680aggcttggag taaaatcagc agatgaccct aaaatactaa atacgctgaa agtcgtggat 1740gaaacaataa aagtcgatac accgaaagga ccatcatggt ataggtataa tcatgatgga 1800tatggtgaga tgtctaagac agaactatat catgggacag gaaaaggaag attgtggcca 1860ctgcttacag gtgagagagg catgtacgaa attgctgcag agtatgatga tgtaataatt 1920ataaagacaa gaataggttt attgaaaggc tcaaggataa gatttgagta cgatatagtg 1980aaagaagatg aaaataagct tttagcacaa ggtatgacag aacacccatt tacgacactt 2040gacagaaaac ctgtaaatat aaaaaagatt ttgcctcatg tttatgaaat gttgaacaaa 2100tgctatgatg atggtgttta g 2121

Thus, an exemplary amplification primer sequence pair is residues 1 to21 of SEQ ID NO:15 (i.e., atgaaattgaatagaaaactt) and the complementarystrand of the last 21 residues of SEQ ID NO:15 (i.e., the complementarystrand of tgctatgatgatggtgtttag).

Amplification reactions can also be used to quantify the amount ofnucleic acid in a sample (such as the amount of message in a cellsample), label the nucleic acid (e.g., to apply it to an array or ablot), detect the nucleic acid, or quantify the amount of a specificnucleic acid in a sample. In one aspect of the invention, messageisolated from a cell or a cDNA library are amplified.

The skilled artisan can select and design suitable oligonucleotideamplification primers. Amplification methods are also well known in theart, and include, e.g., polymerase chain reaction, PCR (see, e.g., PCRPROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, AcademicPress, N.Y. (1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press,Inc., N.Y., ligase chain reaction (LCR) (see, e.g., Wu (1989) Genomics4:560; Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117);transcription amplification (see, e.g., Kwoh (1989) Proc. Natl. Acad.Sci. USA 86:1173); and, self-sustained sequence replication (see, e.g.,Guatelli (1990) Proc. Natl. Acad. Sci. USA 87:1874); Q Beta replicaseamplification (see, e.g., Smith (1997) J. Clin. Microbiol.35:1477-1491), automated Q-beta replicase amplification assay (see,e.g., Burg (1996) Mol. Cell. Probes 10:257-271) and other RNA polymerasemediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario); seealso Berger (1987) Methods Enzymol. 152:307-316; Sambrook; Ausubel; U.S.Pat. Nos. 4,683,195 and 4,683,202; Sooknanan (1995) Biotechnology13:563-564.

Determining the Degree of Sequence Identity

The invention provides nucleic acids having at least 90% sequenceidentity to SEQ ID NO:5, nucleic acids having at least 60% sequenceidentity to SEQ ID NO:7, nucleic acids having at least 50% sequenceidentity to SEQ ID NO:11, nucleic acids having at least 70% sequenceidentity to SEQ ID NO:13, nucleic acids having at least 80% sequenceidentity to SEQ ID NO:15. In one aspect, the invention provides nucleicacids and polypeptides having at least 99%, 98%, 97%, 96%, 95%, 90%,85%, 80%, 75%, 70%, 65%, 60%, 55% or 50% sequence identity (homology) toSEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16. In oneaspect, the invention provides nucleic acids and polypeptide havingsequence as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16. In alternativeaspects, the sequence identity can be over a region of at least about 5,10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1000, or more consecutiveresidues, or the full length of the nucleic acid or polypeptide. Theextent of sequence identity (homology) may be determined using anycomputer program and associated parameters, including those describedherein, such as BLAST 2.2.2. or FASTA version 3.0t78, with the defaultparameters.

Homologous sequences also include RNA sequences in which uridinesreplace the thymines in the nucleic acid sequences. The homologoussequences may be obtained using any of the procedures described hereinor may result from the correction of a sequencing error. It will beappreciated that the nucleic acid sequences as set forth herein can berepresented in the traditional single character format (see, e.g.,Stryer, Lubert. Biochemistry, 3rd Ed., W. H Freeman & Co., New York) orin any other format which records the identity of the nucleotides in asequence.

Various sequence comparison programs identified herein are used in thisaspect of the invention. Protein and/or nucleic acid sequence identities(homologies) may be evaluated using any of the variety of sequencecomparison algorithms and programs known in the art. Such algorithms andprograms include, but are not limited to, TBLASTN, BLASTP, FASTA,TFASTA, and CLUSTALW (Pearson and Lipman, Proc. Natl. Acad. Sci. USA85(8):2444-2448, 1988; Altschul et al., J. Mol. Biol. 215(3):403-410,1990; Thompson et al., Nucleic Acids Res. 22(2):4673-4680, 1994; Higginset al., Methods Enzymol. 266:383-402, 1996; Altschul et al., J. Mol.Biol. 215(3):403-410, 1990; Altschul et al., Nature Genetics 3:266-272,1993).

Homology or identity can be measured using sequence analysis software(e.g., Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705). Such software matches similar sequences byassigning degrees of homology to various deletions, substitutions andother modifications. The terms “homology” and “identity” in the contextof two or more nucleic acids or polypeptide sequences, refer to two ormore sequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same whencompared and aligned for maximum correspondence over a comparison windowor designated region as measured using any number of sequence comparisonalgorithms or by manual alignment and visual inspection. For sequencecomparison, one sequence can act as a reference sequence (a sequence ofthe invention, e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, or SEQ ID NO:16, to which test sequences are compared. When usinga sequence comparison algorithm, test and reference sequences areentered into a computer, subsequence coordinates are designated, ifnecessary, and sequence algorithm program parameters are designated.Default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the numbers of contiguous residues. For example, inalternative aspects of the invention, contiguous residues ranginganywhere from 20 to the full length of an exemplary polypeptide ornucleic acid sequence of the invention are compared to a referencesequence of the same number of contiguous positions after the twosequences are optimally aligned. If the reference sequence has therequisite sequence identity to an exemplary polypeptide or nucleic acidsequence of the invention, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%or 95% sequence identity to a sequence of the invention (including SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ IDNO:16), that sequence is within the scope of the invention. Inalternative embodiments, subsequences ranging from about 20 to 600,about 50 to 200, and about 100 to 150 are compared to a referencesequence of the same number of contiguous positions after the twosequences are optimally aligned. Methods of alignment of sequence forcomparison are well known in the art. Optimal alignment of sequences forcomparison can be conducted, e.g., by the local homology algorithm ofSmith & Waterman, Adv. Appl. Math. 2:482, 1981, by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443, 1970,by the search for similarity method of person & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444, 1988, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection. Other algorithmsfor determining homology or identity include, for example, in additionto a BLAST program (Basic Local Alignment Search Tool at the NationalCenter for Biological Information), ALIGN, AMAS (Analysis of MultiplyAligned Sequences), AMPS (Protein Multiple Sequence Alignment), ASSET(Aligned Segment Statistical Evaluation Tool), BANDS, BESTSCOR, BIOSCAN(Biological Sequence Comparative Analysis Node), BLIMPS (BLocks IMProvedSearcher), FASTA, Intervals & Points, BMB, CLUSTAL V, CLUSTAL W,CONSENSUS, LCONSENSUS, WCONSENSUS, Smith-Waterman algorithm, DARWIN, LasVegas algorithm, FNAT (Forced Nucleotide Alignment Tool), Framealign,Framesearch, DYNAMIC, FILTER, FSAP (Fristensky Sequence AnalysisPackage), GAP (Global Alignment Program), GENAL, GIBBS, GenQuest, ISSC(Sensitive Sequence Comparison), LALIGN (Local Sequence Alignment), LCP(Local Content Program), MACAW (Multiple Alignment Construction &Analysis Workbench), MAP (Multiple Alignment Program), MBLKP, MBLKN,PIMA (Pattern-Induced Multi-sequence Alignment), SAGA (SequenceAlignment by Genetic Algorithm) and WHAT-IF. Such alignment programs canalso be used to screen genome databases to identify polynucleotidesequences having substantially identical sequences. A number of genomedatabases are available, for example, a substantial portion of the humangenome is available as part of the Human Genome Sequencing Project(Gibbs, 1995). Several genomes have been sequenced, e.g., M. genitalium(Fraser et al., 1995), M. jannaschii (Bult et al., 1996), H. influenzae(Fleischmann et al., 1995), E. coli (Blattner et al., 1997), and yeast(S. cerevisiae) (Mewes et al., 1997), and D. melanogaster (Adams et al.,2000). Significant progress has also been made in sequencing the genomesof model organism, such as mouse, C. elegans, and Arabadopsis sp.Databases containing genomic information annotated with some functionalinformation are maintained by different organization, and are accessiblevia the interne.

BLAST, BLAST 2.0 and BLAST 2.2.2 algorithms are also used to practicethe invention. They are described, e.g., in Altschul (1977) Nuc. AcidsRes. 25:3389-3402; Altschul (1990) J. Mol. Biol. 215:403-410. Softwarefor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul (1990) supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are extended in both directions alongeach sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectations (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands. The BLAST algorithm also performs a statisticalanalysis of the similarity between two sequences (see, e.g., Karlin &Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873). One measure ofsimilarity provided by BLAST algorithm is the smallest sum probability(P(N)), which provides an indication of the probability by which a matchbetween two nucleotide or amino acid sequences would occur by chance.For example, a nucleic acid is considered similar to a referencessequence if the smallest sum probability in a comparison of the testnucleic acid to the reference nucleic acid is less than about 0.2, morepreferably less than about 0.01, and most preferably less than about0.001. In one aspect, protein and nucleic acid sequence homologies areevaluated using the Basic Local Alignment Search Tool (“BLAST”). Forexample, five specific BLAST programs can be used to perform thefollowing task: (1) BLASTP and BLAST3 compare an amino acid querysequence against a protein sequence database; (2) BLASTN compares anucleotide query sequence against a nucleotide sequence database; (3)BLASTX compares the six-frame conceptual translation products of a querynucleotide sequence (both strands) against a protein sequence database;(4) TBLASTN compares a query protein sequence against a nucleotidesequence database translated in all six reading frames (both strands);and, (5) TBLASTX compares the six-frame translations of a nucleotidequery sequence against the six-frame translations of a nucleotidesequence database. The BLAST programs identify homologous sequences byidentifying similar segments, which are referred to herein as“high-scoring segment pairs,” between a query amino or nucleic acidsequence and a test sequence which is preferably obtained from a proteinor nucleic acid sequence database. High-scoring segment pairs arepreferably identified (i.e., aligned) by means of a scoring matrix, manyof which are known in the art. Preferably, the scoring matrix used isthe BLOSUM62 matrix (Gonnet et al., Science 256:1443-1445, 1992;Henikoff and Henikoff, Proteins 17:49-61, 1993). Less preferably, thePAM or PAM250 matrices may also be used (see, e.g., Schwartz andDayhoff, eds., 1978, Matrices for Detecting Distance Relationships:Atlas of Protein Sequence and Structure, Washington: National BiomedicalResearch Foundation).

In one aspect of the invention, to determine if a nucleic acid has therequisite sequence identity to be within the scope of the invention, theNCBI BLAST 2.2.2 programs is used, default options to blastp. There areabout 38 setting options in the BLAST 2.2.2 program. In this exemplaryaspect of the invention, all default values are used except for thedefault filtering setting (i.e., all parameters set to default exceptfiltering which is set to OFF); in its place a “-F F” setting is used,which disables filtering. Use of default filtering often results inKarlin-Altschul violations due to short length of sequence.

The default values used in this exemplary aspect of the inventioninclude:

“Filter for low complexity: ON

Word Size: 3

Matrix: Blosum62

Gap Costs Existence: 11

Extension: 1”

Other default settings can be: filter for low complexity OFF, word sizeof 3 for protein, BLOSUM62 matrix, gap existence penalty of −11 and agap extension penalty of −1. An exemplary NCBI BLAST 2.2.2 programsetting has the “-W” option default to 0. This means that, if not set,the word size defaults to 3 for proteins and 11 for nucleotides.

Computer Systems and Computer Program Products

To determine and identify sequence identities, structural homologies,motifs and the like in silico, the sequence of the invention can bestored, recorded, and manipulated on any medium which can be read andaccessed by a computer. Accordingly, the invention provides computers,computer systems, computer readable mediums, computer programs productsand the like recorded or stored thereon the nucleic acid and polypeptidesequences of the invention. As used herein, the words “recorded” and“stored” refer to a process for storing information on a computermedium. A skilled artisan can readily adopt any known methods forrecording information on a computer readable medium to generatemanufactures comprising one or more of the nucleic acid and/orpolypeptide sequences of the invention.

Another aspect of the invention is a computer readable medium havingrecorded thereon at least one nucleic acid and/or polypeptide sequenceof the invention. Computer readable media include magnetically readablemedia, optically readable media, electronically readable media andmagnetic/optical media. For example, the computer readable media may bea hard disk, a floppy disk, a magnetic tape, CD-ROM, Digital VersatileDisk (DVD), Random Access Memory (RAM), or Read Only Memory (ROM) aswell as other types of other media known to those skilled in the art.

Aspects of the invention include systems (e.g., internet based systems),particularly computer systems, which store and manipulate the sequencesand sequence information described herein. One example of a computersystem 100 is illustrated in block diagram form in FIG. 1. As usedherein, “a computer system” refers to the hardware components, softwarecomponents, and data storage components used to analyze a nucleotide orpolypeptide sequence of the invention. The computer system 100 caninclude a processor for processing, accessing and manipulating thesequence data. The processor 105 can be any well-known type of centralprocessing unit, such as, for example, the Pentium III from IntelCorporation, or similar processor from Sun, Motorola, Compaq, AMD orInternational Business Machines. The computer system 100 is a generalpurpose system that comprises the processor 105 and one or more internaldata storage components 110 for storing data, and one or more dataretrieving devices for retrieving the data stored on the data storagecomponents. A skilled artisan can readily appreciate that any one of thecurrently available computer systems are suitable.

In one aspect, the computer system 100 includes a processor 105connected to a bus which is connected to a main memory 115 (preferablyimplemented as RAM) and one or more internal data storage devices 110,such as a hard drive and/or other computer readable media having datarecorded thereon. The computer system 100 can further include one ormore data retrieving device 118 for reading the data stored on theinternal data storage devices 110. The data retrieving device 118 mayrepresent, for example, a floppy disk drive, a compact disk drive, amagnetic tape drive, or a modem capable of connection to a remote datastorage system (e.g., via the internet) etc. In some embodiments, theinternal data storage device 110 is a removable computer readable mediumsuch as a floppy disk, a compact disk, a magnetic tape, etc. containingcontrol logic and/or data recorded thereon. The computer system 100 mayadvantageously include or be programmed by appropriate software forreading the control logic and/or the data from the data storagecomponent once inserted in the data retrieving device. The computersystem 100 includes a display 120 which is used to display output to acomputer user. It should also be noted that the computer system 100 canbe linked to other computer systems 125 a-c in a network or wide areanetwork to provide centralized access to the computer system 100.Software for accessing and processing the nucleotide or amino acidsequences of the invention can reside in main memory 115 duringexecution. In some aspects, the computer system 100 may further comprisea sequence comparison algorithm for comparing a nucleic acid sequence ofthe invention. The algorithm and sequence(s) can be stored on a computerreadable medium. A “sequence comparison algorithm” refers to one or moreprograms which are implemented (locally or remotely) on the computersystem 100 to compare a nucleotide sequence with other nucleotidesequences and/or compounds stored within a data storage means. Forexample, the sequence comparison algorithm may compare the nucleotidesequences of the invention stored on a computer readable medium toreference sequences stored on a computer readable medium to identifyhomologies or structural motifs.

The parameters used with the above algorithms may be adapted dependingon the sequence length and degree of homology studied. In some aspects,the parameters may be the default parameters used by the algorithms inthe absence of instructions from the user. FIG. 2 is a flow diagramillustrating one aspect of a process 200 for comparing a new nucleotideor protein sequence with a database of sequences in order to determinethe homology levels between the new sequence and the sequences in thedatabase. The database of sequences can be a private database storedwithin the computer system 100, or a public database such as GENBANKthat is available through the Internet. The process 200 begins at astart state 201 and then moves to a state 202 wherein the new sequenceto be compared is stored to a memory in a computer system 100. Asdiscussed above, the memory could be any type of memory, including RAMor an internal storage device. The process 200 then moves to a state 204wherein a database of sequences is opened for analysis and comparison.The process 200 then moves to a state 206 wherein the first sequencestored in the database is read into a memory on the computer. Acomparison is then performed at a state 210 to determine if the firstsequence is the same as the second sequence. It is important to notethat this step is not limited to performing an exact comparison betweenthe new sequence and the first sequence in the database. Well-knownmethods are known to those of skill in the art for comparing twonucleotide or protein sequences, even if they are not identical. Forexample, gaps can be introduced into one sequence in order to raise thehomology level between the two tested sequences. The parameters thatcontrol whether gaps or other features are introduced into a sequenceduring comparison are normally entered by the user of the computersystem. Once a comparison of the two sequences has been performed at thestate 210, a determination is made at a decision state 210 whether thetwo sequences are the same. Of course, the term “same” is not limited tosequences that are absolutely identical. Sequences that are within thehomology parameters entered by the user will be marked as “same” in theprocess 200. If a determination is made that the two sequences are thesame, the process 200 moves to a state 214 wherein the name of thesequence from the database is displayed to the user. This state notifiesthe user that the sequence with the displayed name fulfills the homologyconstraints that were entered. Once the name of the stored sequence isdisplayed to the user, the process 200 moves to a decision state 218wherein a determination is made whether more sequences exist in thedatabase. If no more sequences exist in the database, then the process200 terminates at an end state 220. However, if more sequences do existin the database, then the process 200 moves to a state 224 wherein apointer is moved to the next sequence in the database so that it can becompared to the new sequence. In this manner, the new sequence isaligned and compared with every sequence in the database. It should benoted that if a determination had been made at the decision state 212that the sequences were not homologous, then the process 200 would moveimmediately to the decision state 218 in order to determine if any othersequences were available in the database for comparison. Accordingly,one aspect of the invention is a computer system comprising a processor,a data storage device having stored thereon a nucleic acid sequence ofthe invention and a sequence comparer for conducting the comparison. Thesequence comparer may indicate a homology level between the sequencescompared or identify structural motifs, or it may identify structuralmotifs in sequences which are compared to these nucleic acid codes andpolypeptide codes. FIG. 3 is a flow diagram illustrating one embodimentof a process 250 in a computer for determining whether two sequences arehomologous. The process 250 begins at a start state 252 and then movesto a state 254 wherein a first sequence to be compared is stored to amemory. The second sequence to be compared is then stored to a memory ata state 256. The process 250 then moves to a state 260 wherein the firstcharacter in the first sequence is read and then to a state 262 whereinthe first character of the second sequence is read. It should beunderstood that if the sequence is a nucleotide sequence, then thecharacter would normally be either A, T, C, G or U. If the sequence is aprotein sequence, then it can be a single letter amino acid code so thatthe first and sequence sequences can be easily compared. A determinationis then made at a decision state 264 whether the two characters are thesame. If they are the same, then the process 250 moves to a state 268wherein the next characters in the first and second sequences are read.A determination is then made whether the next characters are the same.If they are, then the process 250 continues this loop until twocharacters are not the same. If a determination is made that the nexttwo characters are not the same, the process 250 moves to a decisionstate 274 to determine whether there are any more characters eithersequence to read. If there are not any more characters to read, then theprocess 250 moves to a state 276 wherein the level of homology betweenthe first and second sequences is displayed to the user. The level ofhomology is determined by calculating the proportion of charactersbetween the sequences that were the same out of the total number ofsequences in the first sequence. Thus, if every character in a first 100nucleotide sequence aligned with an every character in a secondsequence, the homology level would be 100%.

Alternatively, the computer program can compare a reference sequence toa sequence of the invention to determine whether the sequences differ atone or more positions. The program can record the length and identity ofinserted, deleted or substituted nucleotides or amino acid residues withrespect to the sequence of either the reference or the invention. Thecomputer program may be a program which determines whether a referencesequence contains a single nucleotide polymorphism (SNP) with respect toa sequence of the invention, or, whether a sequence of the inventioncomprises a SNP of a known sequence. Thus, in some aspects, the computerprogram is a program which identifies SNPs. The method may beimplemented by the computer systems described above and the methodillustrated in FIG. 3. The method can be performed by reading a sequenceof the invention and the reference sequences through the use of thecomputer program and identifying differences with the computer program.

In other aspects the computer based system comprises an identifier foridentifying features within a nucleic acid or polypeptide of theinvention. An “identifier” refers to one or more programs whichidentifies certain features within a nucleic acid sequence. For example,an identifier may comprise a program which identifies an open readingframe (ORF) in a nucleic acid sequence. FIG. 4 is a flow diagramillustrating one aspect of an identifier process 300 for detecting thepresence of a feature in a sequence. The process 300 begins at a startstate 302 and then moves to a state 304 wherein a first sequence that isto be checked for features is stored to a memory 115 in the computersystem 100. The process 300 then moves to a state 306 wherein a databaseof sequence features is opened. Such a database would include a list ofeach feature's attributes along with the name of the feature. Forexample, a feature name could be “Initiation Codon” and the attributewould be “ATG”. Another example would be the feature name “TAATAA Box”and the feature attribute would be “TAATAA”. An example of such adatabase is produced by the University of Wisconsin Genetics ComputerGroup. Alternatively, the features may be structural polypeptide motifssuch as alpha helices, beta sheets, or functional polypeptide motifssuch as enzymatic active sites, helix-turn-helix motifs or other motifsknown to those skilled in the art. Once the database of features isopened at the state 306, the process 300 moves to a state 308 whereinthe first feature is read from the database. A comparison of theattribute of the first feature with the first sequence is then made at astate 310. A determination is then made at a decision state 316 whetherthe attribute of the feature was found in the first sequence. If theattribute was found, then the process 300 moves to a state 318 whereinthe name of the found feature is displayed to the user. The process 300then moves to a decision state 320 wherein a determination is madewhether move features exist in the database. If no more features doexist, then the process 300 terminates at an end state 324. However, ifmore features do exist in the database, then the process 300 reads thenext sequence feature at a state 326 and loops back to the state 310wherein the attribute of the next feature is compared against the firstsequence. If the feature attribute is not found in the first sequence atthe decision state 316, the process 300 moves directly to the decisionstate 320 in order to determine if any more features exist in thedatabase. Thus, in one aspect, the invention provides a computer programthat identifies open reading frames (ORFs).

A polypeptide or nucleic acid sequence of the invention can be storedand manipulated in a variety of data processor programs in a variety offormats. For example, a sequence can be stored as text in a wordprocessing file, such as MicrosoftWORD or WORDPERFECT or as an ASCIIfile in a variety of database programs familiar to those of skill in theart, such as DB2, SYBASE, or ORACLE. In addition, many computer programsand databases may be used as sequence comparison algorithms,identifiers, or sources of reference nucleotide sequences or polypeptidesequences to be compared to a nucleic acid sequence of the invention.The programs and databases used to practice the invention include, butare not limited to: MacPattern (EMBL), DiscoveryBase (MolecularApplications Group), GeneMine (Molecular Applications Group), Look(Molecular Applications Group), MacLook (Molecular Applications Group),BLAST and BLAST2 (NCBI), BLASTN and BLASTX (Altschul et al, J. Mol.Biol. 215: 403, 1990), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci.USA, 85: 2444, 1988), FASTDB (Brutlag et al. Comp. App. Biosci.6:237-245, 1990), Catalyst (Molecular Simulations Inc.), Catalyst/SHAPE(Molecular Simulations Inc.), Cerius2.DBAccess (Molecular SimulationsInc.), HypoGen (Molecular Simulations Inc.), Insight II, (MolecularSimulations Inc.), Discover (Molecular Simulations Inc.), CHARMm(Molecular Simulations Inc.), Felix (Molecular Simulations Inc.),DelPhi, (Molecular Simulations Inc.), QuanteMM, (Molecular SimulationsInc.), Homology (Molecular Simulations Inc.), Modeler (MolecularSimulations Inc.), ISIS (Molecular Simulations Inc.), Quanta/ProteinDesign (Molecular Simulations Inc.), WebLab (Molecular SimulationsInc.), WebLab Diversity Explorer (Molecular Simulations Inc.), GeneExplorer (Molecular Simulations Inc.), SeqFold (Molecular SimulationsInc.), the MDL Available Chemicals Directory database, the MDL Drug DataReport data base, the Comprehensive Medicinal Chemistry database,Derwent's World Drug Index database, the BioByteMasterFile database, theGenbank database, and the Genseqn database. Many other programs and databases would be apparent to one of skill in the art given the presentdisclosure.

Motifs which may be detected using the above programs include sequencesencoding leucine zippers, helix-turn-helix motifs, glycosylation sites,ubiquitination sites, alpha helices, and beta sheets, signal sequencesencoding signal peptides which direct the secretion of the encodedproteins, sequences implicated in transcription regulation such ashomeoboxes, acidic stretches, enzymatic active sites, substrate bindingsites, and enzymatic cleavage sites.

Hybridization of Nucleic Acids

The invention provides isolated or recombinant nucleic acids thathybridize under stringent conditions to an exemplary sequence of theinvention, e.g., a sequence as set forth in SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, orSEQ ID NO:15, or a nucleic acid that encodes a polypeptide of theinvention. The stringent conditions can be highly stringent conditions,medium stringent conditions, low stringent conditions, including thehigh and reduced stringency conditions described herein. In one aspect,it is the stringency of the wash conditions that set forth theconditions which determine whether a nucleic acid is within the scope ofthe invention, as discussed below.

In alternative embodiments, nucleic acids of the invention as defined bytheir ability to hybridize under stringent conditions can be betweenabout five residues and the full length of nucleic acid of theinvention; e.g., they can be at least 5, 10, 15, 20, 25, 30, 35, 40, 50,55, 60, 65, 70, 75, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more, residues inlength. Nucleic acids shorter than full length are also included. Thesenucleic acids can be useful as, e.g., hybridization probes, labelingprobes, PCR oligonucleotide probes, iRNA, antisense or sequencesencoding antibody binding peptides (epitopes), motifs, active sites andthe like.

In one aspect, nucleic acids of the invention are defined by theirability to hybridize under high stringency comprises conditions of about50% formamide at about 37° C. to 42° C. In one aspect, nucleic acids ofthe invention are defined by their ability to hybridize under reducedstringency comprising conditions in about 35% to 25% formamide at about30° C. to 35° C.

Alternatively, nucleic acids of the invention are defined by theirability to hybridize under high stringency comprising conditions at 42°C. in 50% formamide, 5×SSPE, 0.3% SDS, and a repetitive sequenceblocking nucleic acid, such as cot-1 or salmon sperm DNA (e.g., 200 n/mlsheared and denatured salmon sperm DNA). In one aspect, nucleic acids ofthe invention are defined by their ability to hybridize under reducedstringency conditions comprising 35% formamide at a reduced temperatureof 35° C.

Following hybridization, the filter may be washed with 6×SSC, 0.5% SDSat 50° C. These conditions are considered to be “moderate” conditionsabove 25% formamide and “low” conditions below 25% formamide. A specificexample of “moderate” hybridization conditions is when the abovehybridization is conducted at 30% formamide. A specific example of “lowstringency” hybridization conditions is when the above hybridization isconducted at 10% formamide.

The temperature range corresponding to a particular level of stringencycan be further narrowed by calculating the purine to pyrimidine ratio ofthe nucleic acid of interest and adjusting the temperature accordingly.Nucleic acids of the invention are also defined by their ability tohybridize under high, medium, and low stringency conditions as set forthin Ausubel and Sambrook. Variations on the above ranges and conditionsare well known in the art. Hybridization conditions are discussedfurther, below.

The above procedure may be modified to identify nucleic acids havingdecreasing levels of homology to the probe sequence. For example, toobtain nucleic acids of decreasing homology to the detectable probe,less stringent conditions may be used. For example, the hybridizationtemperature may be decreased in increments of 5° C. from 68° C. to 42°C. in a hybridization buffer having a Na⁺ concentration of approximately1M. Following hybridization, the filter may be washed with 2×SSC, 0.5%SDS at the temperature of hybridization. These conditions are consideredto be “moderate” conditions above 50° C. and “low” conditions below 50°C. A specific example of “moderate” hybridization conditions is when theabove hybridization is conducted at 55° C. A specific example of “lowstringency” hybridization conditions is when the above hybridization isconducted at 45° C.

Alternatively, the hybridization may be carried out in buffers, such as6×SSC, containing formamide at a temperature of 42° C. In this case, theconcentration of formamide in the hybridization buffer may be reduced in5% increments from 50% to 0% to identify clones having decreasing levelsof homology to the probe. Following hybridization, the filter may bewashed with 6×SSC, 0.5% SDS at 50° C. These conditions are considered tobe “moderate” conditions above 25% formamide and “low” conditions below25% formamide. A specific example of “moderate” hybridization conditionsis when the above hybridization is conducted at 30% formamide. Aspecific example of “low stringency” hybridization conditions is whenthe above hybridization is conducted at 10% formamide.

However, the selection of a hybridization format is not critical—it isthe stringency of the wash conditions that set forth the conditionswhich determine whether a nucleic acid is within the scope of theinvention. Wash conditions used to identify nucleic acids within thescope of the invention include, e.g.: a salt concentration of about 0.02molar at pH 7 and a temperature of at least about 50° C. or about 55° C.to about 60° C.; or, a salt concentration of about 0.15 M NaCl at 72° C.for about 15 minutes; or, a salt concentration of about 0.2×SSC at atemperature of at least about 50° C. or about 55° C. to about 60° C. forabout 15 to about 20 minutes; or, the hybridization complex is washedtwice with a solution with a salt concentration of about 2×SSCcontaining 0.1% SDS at room temperature for 15 minutes and then washedtwice by 0.1×SSC containing 0.1% SDS at 68° C. for 15 minutes; or,equivalent conditions. See Sambrook, Tijssen and Ausubel for adescription of SSC buffer and equivalent conditions.

These methods may be used to isolate nucleic acids of the invention.

Oligonucleotides Probes and Methods for Using Them

The invention also provides nucleic acid probes that can be used, e.g.,for identifying nucleic acids encoding a polypeptide with an amylaseactivity or fragments thereof or for identifying amylase genes. In oneaspect, the probe comprises at least 10 consecutive bases of a nucleicacid of the invention. Alternatively, a probe of the invention can be atleast about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70,80, 90, 100, 110, 120, 130, 150 or about 10 to 50, about 20 to 60 about30 to 70, consecutive bases of a sequence as set forth in a nucleic acidof the invention. The probes identify a nucleic acid by binding and/orhybridization. The probes can be used in arrays of the invention, seediscussion below, including, e.g., capillary arrays. The probes of theinvention can also be used to isolate other nucleic acids orpolypeptides.

The probes of the invention can be used to determine whether abiological sample, such as a soil sample, contains an organism having anucleic acid sequence of the invention or an organism from which thenucleic acid was obtained. In such procedures, a biological samplepotentially harboring the organism from which the nucleic acid wasisolated is obtained and nucleic acids are obtained from the sample. Thenucleic acids are contacted with the probe under conditions which permitthe probe to specifically hybridize to any complementary sequencespresent in the sample. Where necessary, conditions which permit theprobe to specifically hybridize to complementary sequences may bedetermined by placing the probe in contact with complementary sequencesfrom samples known to contain the complementary sequence, as well ascontrol sequences which do not contain the complementary sequence.Hybridization conditions, such as the salt concentration of thehybridization buffer, the formamide concentration of the hybridizationbuffer, or the hybridization temperature, may be varied to identifyconditions which allow the probe to hybridize specifically tocomplementary nucleic acids (see discussion on specific hybridizationconditions).

If the sample contains the organism from which the nucleic acid wasisolated, specific hybridization of the probe is then detected.Hybridization may be detected by labeling the probe with a detectableagent such as a radioactive isotope, a fluorescent dye or an enzymecapable of catalyzing the formation of a detectable product. Manymethods for using the labeled probes to detect the presence ofcomplementary nucleic acids in a sample are familiar to those skilled inthe art. These include Southern Blots, Northern Blots, colonyhybridization procedures, and dot blots. Protocols for each of theseprocedures are provided in Ausubel and Sambrook.

Alternatively, more than one probe (at least one of which is capable ofspecifically hybridizing to any complementary sequences which arepresent in the nucleic acid sample), may be used in an amplificationreaction to determine whether the sample contains an organism containinga nucleic acid sequence of the invention (e.g., an organism from whichthe nucleic acid was isolated). In one aspect, the probes compriseoligonucleotides. In one aspect, the amplification reaction may comprisea PCR reaction. PCR protocols are described in Ausubel and Sambrook (seediscussion on amplification reactions). In such procedures, the nucleicacids in the sample are contacted with the probes, the amplificationreaction is performed, and any resulting amplification product isdetected. The amplification product may be detected by performing gelelectrophoresis on the reaction products and staining the gel with anintercalator such as ethidium bromide. Alternatively, one or more of theprobes may be labeled with a radioactive isotope and the presence of aradioactive amplification product may be detected by autoradiographyafter gel electrophoresis.

Probes derived from sequences near the 3′ or 5′ ends of a nucleic acidsequence of the invention can also be used in chromosome walkingprocedures to identify clones containing additional, e.g., genomicsequences. Such methods allow the isolation of genes which encodeadditional proteins of interest from the host organism.

In one aspect, nucleic acid sequences of the invention are used asprobes to identify and isolate related nucleic acids. In some aspects,the so-identified related nucleic acids may be cDNAs or genomic DNAsfrom organisms other than the one from which the nucleic acid of theinvention was first isolated. In such procedures, a nucleic acid sampleis contacted with the probe under conditions which permit the probe tospecifically hybridize to related sequences. Hybridization of the probeto nucleic acids from the related organism is then detected using any ofthe methods described above.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency can vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (e.g., GC v. ATcontent), and nucleic acid type (e.g., RNA v. DNA) of the hybridizingregions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.Hybridization can be carried out under conditions of low stringency,moderate stringency or high stringency. As an example of nucleic acidhybridization, a polymer membrane containing immobilized denaturednucleic acids is first prehybridized for 30 minutes at 45° C. in asolution consisting of 0.9 M NaCl, 50 mM NaH₂PO4, pH 7.0, 5.0 mMNa₂EDTA, 0.5% SDS, 10×Denhardt's, and 0.5 mg/ml polyriboadenylic acid.Approximately 2×10⁷ cpm (specific activity 4-9×10⁸ cpm/ug) of ³²Pend-labeled oligonucleotide probe can then added to the solution. After12-16 hours of incubation, the membrane is washed for 30 minutes at roomtemperature (RT) in 1×SET (150 mM NaCl, 20 mM Tris hydrochloride, pH7.8, 1 mM Na₂EDTA) containing 0.5% SDS, followed by a 30 minute wash infresh 1×SET at Tm-10° C. for the oligonucleotide probe. The membrane isthen exposed to auto-radiographic film for detection of hybridizationsignals.

By varying the stringency of the hybridization conditions used toidentify nucleic acids, such as cDNAs or genomic DNAs, which hybridizeto the detectable probe, nucleic acids having different levels ofhomology to the probe can be identified and isolated. Stringency may bevaried by conducting the hybridization at varying temperatures below themelting temperatures of the probes. The melting temperature, Tm, is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly complementary probe. Verystringent conditions are selected to be equal to or about 5° C. lowerthan the Tm for a particular probe. The melting temperature of the probemay be calculated using the following exemplary formulas. For probesbetween 14 and 70 nucleotides in length the melting temperature (Tm) iscalculated using the formula: Tm=81.5+16.6(log [Na+])+0.41(fractionG+C)−(600/N) where N is the length of the probe. If the hybridization iscarried out in a solution containing formamide, the melting temperaturemay be calculated using the equation: Tm=81.5+16.6(log[Na+])+0.41(fraction G+C)−(0.63% formamide)−(600/N) where N is thelength of the probe. Prehybridization may be carried out in 6×SSC,5×Denhardt's reagent, 0.5% SDS, 100 μg denatured fragmented salmon spermDNA or 6×SSC, 5×Denhardt's reagent, 0.5% SDS, 100 μg denaturedfragmented salmon sperm DNA, 50% formamide. Formulas for SSC andDenhardt's and other solutions are listed, e.g., in Sambrook.

Hybridization is conducted by adding the detectable probe to theprehybridization solutions listed above. Where the probe comprisesdouble stranded DNA, it is denatured before addition to thehybridization solution. The filter is contacted with the hybridizationsolution for a sufficient period of time to allow the probe to hybridizeto cDNAs or genomic DNAs containing sequences complementary thereto orhomologous thereto. For probes over 200 nucleotides in length, thehybridization may be carried out at 15-25° C. below the Tm. For shorterprobes, such as oligonucleotide probes, the hybridization may beconducted at 5-10° C. below the Tm. In one aspect, hybridizations in6×SSC are conducted at approximately 68° C. In one aspect,hybridizations in 50% formamide containing solutions are conducted atapproximately 42° C. All of the foregoing hybridizations would beconsidered to be under conditions of high stringency.

Following hybridization, the filter is washed to remove anynon-specifically bound detectable probe. The stringency used to wash thefilters can also be varied depending on the nature of the nucleic acidsbeing hybridized, the length of the nucleic acids being hybridized, thedegree of complementarity, the nucleotide sequence composition (e.g., GCv. AT content), and the nucleic acid type (e.g., RNA v. DNA). Examplesof progressively higher stringency condition washes are as follows:2×SSC, 0.1% SDS at room temperature for 15 minutes (low stringency);0.1×SSC, 0.5% SDS at room temperature for 30 minutes to 1 hour (moderatestringency); 0.1×SSC, 0.5% SDS for 15 to 30 minutes at between thehybridization temperature and 68° C. (high stringency); and 0.15M NaClfor 15 minutes at 72° C. (very high stringency). A final low stringencywash can be conducted in 0.1×SSC at room temperature. The examples aboveare merely illustrative of one set of conditions that can be used towash filters. One of skill in the art would know that there are numerousrecipes for different stringency washes.

Nucleic acids which have hybridized to the probe can be identified byautoradiography or other conventional techniques. The above proceduremay be modified to identify nucleic acids having decreasing levels ofhomology to the probe sequence. For example, to obtain nucleic acids ofdecreasing homology to the detectable probe, less stringent conditionsmay be used. For example, the hybridization temperature may be decreasedin increments of 5° C. from 68° C. to 42° C. in a hybridization bufferhaving a Na⁺ concentration of approximately 1M. Following hybridization,the filter may be washed with 2×SSC, 0.5% SDS at the temperature ofhybridization. These conditions are considered to be “moderate”conditions above 50° C. and “low” conditions below 50° C. An example of“moderate” hybridization conditions is when the above hybridization isconducted at 55° C. An example of “low stringency” hybridizationconditions is when the above hybridization is conducted at 45° C.

Alternatively, the hybridization may be carried out in buffers, such as6×SSC, containing formamide at a temperature of 42° C. In this case, theconcentration of formamide in the hybridization buffer may be reduced in5% increments from 50% to 0% to identify clones having decreasing levelsof homology to the probe. Following hybridization, the filter may bewashed with 6×SSC, 0.5% SDS at 50° C. These conditions are considered tobe “moderate” conditions above 25% formamide and “low” conditions below25% formamide. A specific example of “moderate” hybridization conditionsis when the above hybridization is conducted at 30% formamide. Aspecific example of “low stringency” hybridization conditions is whenthe above hybridization is conducted at 10% formamide.

These probes and methods of the invention can be used to isolate nucleicacids having a sequence with at least about 99%, 98%, 97%, at least 95%,at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, atleast 65%, at least 60%, at least 55%, or at least 50% homology to anucleic acid sequence of the invention comprising at least about 10, 15,20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1000, or more consecutive basesthereof, and the sequences complementary thereto. Homology may bemeasured using an alignment algorithm, as discussed herein. For example,the homologous polynucleotides may have a coding sequence which is anaturally occurring allelic variant of one of the coding sequencesdescribed herein. Such allelic variants may have a substitution,deletion or addition of one or more nucleotides when compared to anucleic acid of the invention.

Additionally, the probes and methods of the invention can be used toisolate nucleic acids which encode polypeptides having at least about99%, at least 95%, at least 90%, at least 85%, at least 80%, at least75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least50% sequence identity (homology) to a polypeptide of the inventioncomprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids, as determined using a sequence alignmentalgorithm (e.g., such as the FASTA version 3.0t78 algorithm with thedefault parameters, or a BLAST 2.2.2 program with exemplary settings asset forth herein).

Inhibiting Expression of Amylase

The invention provides nucleic acids complementary to (e.g., antisensesequences to) the nucleic acid sequences of the invention. Antisensesequences are capable of inhibiting the transport, splicing ortranscription of amylase-encoding genes. The inhibition can be effectedthrough the targeting of genomic DNA or messenger RNA. The transcriptionor function of targeted nucleic acid can be inhibited, for example, byhybridization and/or cleavage. One particularly useful set of inhibitorsprovided by the present invention includes oligonucleotides which areable to either bind amylase gene or message, in either case preventingor inhibiting the production or function of amylase. The association canbe through sequence specific hybridization. Another useful class ofinhibitors includes oligonucleotides which cause inactivation orcleavage of amylase message. The oligonucleotide can have enzymeactivity which causes such cleavage, such as ribozymes. Theoligonucleotide can be chemically modified or conjugated to an enzyme orcomposition capable of cleaving the complementary nucleic acid. A poolof many different such oligonucleotides can be screened for those withthe desired activity. Thus, the invention provides various compositionsfor the inhibition of protease expression on a nucleic acid and/orprotein level, e.g., antisense, iRNA and ribozymes comprising proteasesequences of the invention and the anti-protease antibodies of theinvention.

Inhibition of amylase expression can have a variety of industrialapplications. For example, inhibition of amylase expression can slow orprevent spoilage. Spoilage can occur when polysaccharides, e.g.,structural polysaccharides, are enzymatically degraded. This can lead tothe deterioration, or rot, of fruits and vegetables. In one aspect, useof compositions of the invention that inhibit the expression and/oractivity of amylases, e.g., antibodies, antisense oligonucleotides,ribozymes and RNAi, are used to slow or prevent spoilage. Thus, in oneaspect, the invention provides methods and compositions comprisingapplication onto a plant or plant product (e.g., a fruit, seed, root,leaf, etc.) antibodies, antisense oligonucleotides, ribozymes and RNAiof the invention to slow or prevent spoilage. These compositions alsocan be expressed by the plant (e.g., a transgenic plant) or anotherorganism (e.g., a bacterium or other microorganism transformed with anamylase gene of the invention).

The compositions of the invention for the inhibition of amylaseexpression (e.g., antisense, iRNA, ribozymes, antibodies) can be used aspharmaceutical compositions, e.g., as anti-pathogen agents or in othertherapies, e.g., anti-inflammatory or skin or digestive aid treatments.

Antisense Oligonucleotides

The invention provides antisense oligonucleotides capable of bindingamylase message which can inhibit proteolytic activity by targetingmRNA. Strategies for designing antisense oligonucleotides are welldescribed in the scientific and patent literature, and the skilledartisan can design such amylase oligonucleotides using the novelreagents of the invention. For example, gene walking/RNA mappingprotocols to screen for effective antisense oligonucleotides are wellknown in the art, see, e.g., Ho (2000) Methods Enzymol. 314:168-183,describing an RNA mapping assay, which is based on standard moleculartechniques to provide an easy and reliable method for potent antisensesequence selection. See also Smith (2000) Eur. J. Pharm. Sci.11:191-198.

Naturally occurring nucleic acids are used as antisenseoligonucleotides. The antisense oligonucleotides can be of any length;for example, in alternative aspects, the antisense oligonucleotides arebetween about 5 to 100, about 10 to 80, about 15 to 60, about 18 to 40.The optimal length can be determined by routine screening. The antisenseoligonucleotides can be present at any concentration. The optimalconcentration can be determined by routine screening. A wide variety ofsynthetic, non-naturally occurring nucleotide and nucleic acid analoguesare known which can address this potential problem. For example, peptidenucleic acids (PNAs) containing non-ionic backbones, such asN-(2-aminoethyl) glycine units can be used. Antisense oligonucleotideshaving phosphorothioate linkages can also be used, as described in WO97/03211; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol 144:189-197;Antisense Therapeutics, ed. Agrawal (Humana Press, Totowa, N.J., 1996).Antisense oligonucleotides having synthetic DNA backbone analoguesprovided by the invention can also include phosphoro-dithioate,methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate,3′-thioacetal, methylene(methylimino), 3′-N-carbamate, and morpholinocarbamate nucleic acids, as described above.

Combinatorial chemistry methodology can be used to create vast numbersof oligonucleotides that can be rapidly screened for specificoligonucleotides that have appropriate binding affinities andspecificities toward any target, such as the sense and antisense amylasesequences of the invention (see, e.g., Gold (1995) J. of Biol. Chem.270:13581-13584).

Inhibitory Ribozymes

The invention provides ribozymes capable of binding amylase message.These ribozymes can inhibit amylase activity by, e.g., targeting mRNA.Strategies for designing ribozymes and selecting the amylase-specificantisense sequence for targeting are well described in the scientificand patent literature, and the skilled artisan can design such ribozymesusing the novel reagents of the invention. Ribozymes act by binding to atarget RNA through the target RNA binding portion of a ribozyme which isheld in close proximity to an enzymatic portion of the RNA that cleavesthe target RNA. Thus, the ribozyme recognizes and binds a target RNAthrough complementary base-pairing, and once bound to the correct site,acts enzymatically to cleave and inactivate the target RNA. Cleavage ofa target RNA in such a manner will destroy its ability to directsynthesis of an encoded protein if the cleavage occurs in the codingsequence. After a ribozyme has bound and cleaved its RNA target, it canbe released from that RNA to bind and cleave new targets repeatedly.

In some circumstances, the enzymatic nature of a ribozyme can beadvantageous over other technologies, such as antisense technology(where a nucleic acid molecule simply binds to a nucleic acid target toblock its transcription, translation or association with anothermolecule) as the effective concentration of ribozyme necessary to effecta therapeutic treatment can be lower than that of an antisenseoligonucleotide. This potential advantage reflects the ability of theribozyme to act enzymatically. Thus, a single ribozyme molecule is ableto cleave many molecules of target RNA. In addition, a ribozyme istypically a highly specific inhibitor, with the specificity ofinhibition depending not only on the base pairing mechanism of binding,but also on the mechanism by which the molecule inhibits the expressionof the RNA to which it binds. That is, the inhibition is caused bycleavage of the RNA target and so specificity is defined as the ratio ofthe rate of cleavage of the targeted RNA over the rate of cleavage ofnon-targeted RNA. This cleavage mechanism is dependent upon factorsadditional to those involved in base pairing. Thus, the specificity ofaction of a ribozyme can be greater than that of antisenseoligonucleotide binding the same RNA site.

The ribozyme of the invention, e.g., an enzymatic ribozyme RNA molecule,can be formed in a hammerhead motif, a hairpin motif, as a hepatitisdelta virus motif, a group I intron motif and/or an RNaseP-like RNA inassociation with an RNA guide sequence. Examples of hammerhead motifsare described by, e.g., Rossi (1992) Aids Research and HumanRetroviruses 8:183; hairpin motifs by Hampel (1989) Biochemistry28:4929, and Hampel (1990) Nuc. Acids Res. 18:299; the hepatitis deltavirus motif by Perrotta (1992) Biochemistry 31:16; the RNaseP motif byGuerrier-Takada (1983) Cell 35:849; and the group I intron by Cech U.S.Pat. No. 4,987,071. The recitation of these specific motifs is notintended to be limiting. Those skilled in the art will recognize that aribozyme of the invention, e.g., an enzymatic RNA molecule of thisinvention, can have a specific substrate binding site complementary toone or more of the target gene RNA regions. A ribozyme of the inventioncan have a nucleotide sequence within or surrounding that substratebinding site which imparts an RNA cleaving activity to the molecule.

RNA Interference (RNAi)

In one aspect, the invention provides an RNA inhibitory molecule, aso-called “RNAi” molecule, comprising an amylase sequence of theinvention. The RNAi molecule comprises a double-stranded RNA (dsRNA)molecule. The RNAi can inhibit expression of an amylase gene. In oneaspect, the RNAi is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 ormore duplex nucleotides in length. While the invention is not limited byany particular mechanism of action, the RNAi can enter a cell and causethe degradation of a single-stranded RNA (ssRNA) of similar or identicalsequences, including endogenous mRNAs. When a cell is exposed todouble-stranded RNA (dsRNA), mRNA from the homologous gene isselectively degraded by a process called RNA interference (RNAi). Apossible basic mechanism behind RNAi is the breaking of adouble-stranded RNA (dsRNA) matching a specific gene sequence into shortpieces called short interfering RNA, which trigger the degradation ofmRNA that matches its sequence. In one aspect, the RNAi's of theinvention are used in gene-silencing therapeutics, see, e.g., Shuey(2002) Drug Discov. Today 7:1040-1046. In one aspect, the inventionprovides methods to selectively degrade RNA using the RNAi's of theinvention. The process may be practiced in vitro, ex vivo or in vivo. Inone aspect, the RNAi molecules of the invention can be used to generatea loss-of-function mutation in a cell, an organ or an animal. Methodsfor making and using RNAi molecules for selectively degrade RNA are wellknown in the art, see, e.g., U.S. Pat. Nos. 6,506,559; 6,511,824;6,515,109; 6,489,127.

Modification of Nucleic Acids

The invention provides methods of generating variants of the nucleicacids of the invention, e.g., those encoding am amylase. These methodscan be repeated or used in various combinations to generate amylaseshaving an altered or different activity or an altered or differentstability from that of an amylase encoded by the template nucleic acid.These methods also can be repeated or used in various combinations,e.g., to generate variations in gene/message expression, messagetranslation or message stability. In another aspect, the geneticcomposition of a cell is altered by, e.g., modification of a homologousgene ex vivo, followed by its reinsertion into the cell.

A nucleic acid of the invention can be altered by any means. Forexample, random or stochastic methods, or, non-stochastic, or “directedevolution,” methods, see, e.g., U.S. Pat. No. 6,361,974. Methods forrandom mutation of genes are well known in the art, see, e.g., U.S. Pat.No. 5,830,696. For example, mutagens can be used to randomly mutate agene. Mutagens include, e.g., ultraviolet light or gamma irradiation, ora chemical mutagen, e.g., mitomycin, nitrous acid, photoactivatedpsoralens, alone or in combination, to induce DNA breaks amenable torepair by recombination. Other chemical mutagens include, for example,sodium bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid.Other mutagens are analogues of nucleotide precursors, e.g.,nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. Theseagents can be added to a PCR reaction in place of the nucleotideprecursor thereby mutating the sequence. Intercalating agents such asproflavine, acriflavine, quinacrine and the like can also be used.

Any technique in molecular biology can be used, e.g., random PCRmutagenesis, see, e.g., Rice (1992) Proc. Natl. Acad. Sci. USA89:5467-5471; or, combinatorial multiple cassette mutagenesis, see,e.g., Crameri (1995) Biotechniques 18:194-196. Alternatively, nucleicacids, e.g., genes, can be reassembled after random, or “stochastic,”fragmentation, see, e.g., U.S. Pat. Nos. 6,291,242; 6,287,862;6,287,861; 5,955,358; 5,830,721; 5,824,514; 5,811,238; 5,605,793. Inalternative aspects, modifications, additions or deletions areintroduced by error-prone PCR, shuffling, oligonucleotide-directedmutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis,cassette mutagenesis, recursive ensemble mutagenesis, exponentialensemble mutagenesis, site-specific mutagenesis, gene reassembly, genesite saturated mutagenesis (GSSM), synthetic ligation reassembly (SLR),recombination, recursive sequence recombination, phosphothioate-modifiedDNA mutagenesis, uracil-containing template mutagenesis, gapped duplexmutagenesis, point mismatch repair mutagenesis, repair-deficient hoststrain mutagenesis, chemical mutagenesis, radiogenic mutagenesis,deletion mutagenesis, restriction-selection mutagenesis,restriction-purification mutagenesis, artificial gene synthesis,ensemble mutagenesis, chimeric nucleic acid multimer creation, and/or acombination of these and other methods.

The following publications describe a variety of recursive recombinationprocedures and/or methods which can be incorporated into the methods ofthe invention: Stemmer (1999) “Molecular breeding of viruses fortargeting and other clinical properties” Tumor Targeting 4:1-4; Ness(1999) Nature Biotechnology 17:893-896; Chang (1999) “Evolution of acytokine using DNA family shuffling” Nature Biotechnology 17:793-797;Minshull (1999) “Protein evolution by molecular breeding” CurrentOpinion in Chemical Biology 3:284-290; Christians (1999) “Directedevolution of thymidine kinase for AZT phosphorylation using DNA familyshuffling” Nature Biotechnology 17:259-264; Crameri (1998) “DNAshuffling of a family of genes from diverse species accelerates directedevolution” Nature 391:288-291; Crameri (1997) “Molecular evolution of anarsenate detoxification pathway by DNA shuffling,” Nature Biotechnology15:436-438; Zhang (1997) “Directed evolution of an effective fucosidasefrom a galactosidase by DNA shuffling and screening” Proc. Natl. Acad.Sci. USA 94:4504-4509; Patten et al. (1997) “Applications of DNAShuffling to Pharmaceuticals and Vaccines” Current Opinion inBiotechnology 8:724-733; Crameri et al. (1996) “Construction andevolution of antibody-phage libraries by DNA shuffling” Nature Medicine2:100-103; Gates et al. (1996) “Affinity selective isolation of ligandsfrom peptide libraries through display on a lac repressor ‘headpiecedimer’” Journal of Molecular Biology 255:373-386; Stemmer (1996) “SexualPCR and Assembly PCR” In: The Encyclopedia of Molecular Biology. VCHPublishers, New York. pp. 447-457; Crameri and Stemmer (1995)“Combinatorial multiple cassette mutagenesis creates all thepermutations of mutant and wildtype cassettes” BioTechniques 18:194-195;Stemmer et al. (1995) “Single-step assembly of a gene and entire plasmidform large numbers of oligodeoxyribonucleotides” Gene, 164:49-53;Stemmer (1995) “The Evolution of Molecular Computation” Science 270:1510; Stemmer (1995) “Searching Sequence Space” Bio/Technology13:549-553; Stemmer (1994) “Rapid evolution of a protein in vitro by DNAshuffling” Nature 370:389-391; and Stemmer (1994) “DNA shuffling byrandom fragmentation and reassembly: In vitro recombination formolecular evolution.” Proc. Natl. Acad. Sci. USA 91:10747-10751.

Mutational methods of generating diversity include, for example,site-directed mutagenesis (Ling et al. (1997) “Approaches to DNAmutagenesis: an overview” Anal Biochem. 254(2): 157-178; Dale et al.(1996) “Oligonucleotide-directed random mutagenesis using thephosphorothioate method” Methods Mol. Biol. 57:369-374; Smith (1985) “Invitro mutagenesis” Ann. Rev. Genet. 19:423-462; Botstein & Shortle(1985) “Strategies and applications of in vitro mutagenesis” Science229:1193-1201; Carter (1986) “Site-directed mutagenesis” Biochem. J.237:1-7; and Kunkel (1987) “The efficiency of oligonucleotide directedmutagenesis” in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin)); mutagenesis usinguracil containing templates (Kunkel (1985) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Proc. Natl.Acad. Sci. USA 82:488-492; Kunkel et al. (1987) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Methods inEnzymol. 154, 367-382; and Bass et al. (1988) “Mutant Trp repressorswith new DNA-binding specificities” Science 242:240-245);oligonucleotide-directed mutagenesis (Methods in Enzymol. 100: 468-500(1983); Methods in Enzymol. 154: 329-350 (1987); Zoller & Smith (1982)“Oligonucleotide-directed mutagenesis using M13-derived vectors: anefficient and general procedure for the production of point mutations inany DNA fragment” Nucleic Acids Res. 10:6487-6500; Zoller & Smith (1983)“Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors” Methods in Enzymol. 100:468-500; and Zoller & Smith (1987)Oligonucleotide-directed mutagenesis: a simple method using twooligonucleotide primers and a single-stranded DNA template” Methods inEnzymol. 154:329-350); phosphorothioate-modified DNA mutagenesis (Tayloret al. (1985) “The use of phosphorothioate-modified DNA in restrictionenzyme reactions to prepare nicked DNA” Nucl. Acids Res. 13: 8749-8764;Taylor et al. (1985) “The rapid generation of oligonucleotide-directedmutations at high frequency using phosphorothioate-modified DNA” Nucl.Acids Res. 13: 8765-8787 (1985); Nakamaye (1986) “Inhibition ofrestriction endonuclease Nci I cleavage by phosphorothioate groups andits application to oligonucleotide-directed mutagenesis” Nucl. AcidsRes. 14: 9679-9698; Sayers et al. (1988) “Y-T Exonucleases inphosphorothioate-based oligonucleotide-directed mutagenesis” Nucl. AcidsRes. 16:791-802; and Sayers et al. (1988) “Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide” Nucl. Acids Res. 16:803-814); mutagenesis using gapped duplex DNA (Kramer et al. (1984) “Thegapped duplex DNA approach to oligonucleotide-directed mutationconstruction” Nucl. Acids Res. 12: 9441-9456; Kramer & Fritz (1987)Methods in Enzymol. “Oligonucleotide-directed construction of mutationsvia gapped duplex DNA” 154:350-367; Kramer et al. (1988) “Improvedenzymatic in vitro reactions in the gapped duplex DNA approach tooligonucleotide-directed construction of mutations” Nucl. Acids Res. 16:7207; and Fritz et al. (1988) “Oligonucleotide-directed construction ofmutations: a gapped duplex DNA procedure without enzymatic reactions invitro” Nucl. Acids Res. 16: 6987-6999).

Additional protocols that can be used to practice the invention includepoint mismatch repair (Kramer (1984) “Point Mismatch Repair” Cell38:879-887), mutagenesis using repair-deficient host strains (Carter etal. (1985) “Improved oligonucleotide site-directed mutagenesis using M13vectors” Nucl. Acids Res. 13: 4431-4443; and Carter (1987) “Improvedoligonucleotide-directed mutagenesis using M13 vectors” Methods inEnzymol. 154: 382-403), deletion mutagenesis (Eghtedarzadeh (1986) “Useof oligonucleotides to generate large deletions” Nucl. Acids Res. 14:5115), restriction-selection and restriction-selection andrestriction-purification (Wells et al. (1986) “Importance ofhydrogen-bond formation in stabilizing the transition state ofsubtilisin” Phil. Trans. R. Soc. Lond. A 317: 415-423), mutagenesis bytotal gene synthesis (Nambiar et al. (1984) “Total synthesis and cloningof a gene coding for the ribonuclease S protein” Science 223: 1299-1301;Sakamar and Khorana (1988) “Total synthesis and expression of a gene forthe a-subunit of bovine rod outer segment guanine nucleotide-bindingprotein (transducin)” Nucl. Acids Res. 14: 6361-6372; Wells et al.(1985) “Cassette mutagenesis: an efficient method for generation ofmultiple mutations at defined sites” Gene 34:315-323; and Grundstrom etal. (1985) “Oligonucleotide-directed mutagenesis by microscale‘shot-gun’ gene synthesis” Nucl. Acids Res. 13: 3305-3316),double-strand break repair (Mandecki (1986); Arnold (1993) “Proteinengineering for unusual environments” Current Opinion in Biotechnology4:450-455. “Oligonucleotide-directed double-strand break repair inplasmids of Escherichia coli: a method for site-specific mutagenesis”Proc. Natl. Acad. Sci. USA, 83:7177-7181). Additional details on many ofthe above methods can be found in Methods in Enzymology Volume 154,which also describes useful controls for trouble-shooting problems withvarious mutagenesis methods.

Protocols that can be used to practice the invention are described,e.g., in U.S. Pat. No. 5,605,793 to Stemmer (Feb. 25, 1997), “Methodsfor In Vitro Recombination;” U.S. Pat. No. 5,811,238 to Stemmer et al.(Sep. 22, 1998) “Methods for Generating Polynucleotides having DesiredCharacteristics by Iterative Selection and Recombination;” U.S. Pat. No.5,830,721 to Stemmer et al. (Nov. 3, 1998), “DNA Mutagenesis by RandomFragmentation and Reassembly;” U.S. Pat. No. 5,834,252 to Stemmer, etal. (Nov. 10, 1998) “End-Complementary Polymerase Reaction;” U.S. Pat.No. 5,837,458 to Minshull, et al. (Nov. 17, 1998), “Methods andCompositions for Cellular and Metabolic Engineering;” WO 95/22625,Stemmer and Crameri, “Mutagenesis by Random Fragmentation andReassembly;” WO 96/33207 by Stemmer and Lipschutz “End ComplementaryPolymerase Chain Reaction;” WO 97/20078 by Stemmer and Crameri “Methodsfor Generating Polynucleotides having Desired Characteristics byIterative Selection and Recombination;” WO 97/35966 by Minshull andStemmer, “Methods and Compositions for Cellular and MetabolicEngineering;” WO 99/41402 by Punnonen et al. “Targeting of GeneticVaccine Vectors;” WO 99/41383 by Punnonen et al. “Antigen LibraryImmunization;” WO 99/41369 by Punnonen et al. “Genetic Vaccine VectorEngineering;” WO 99/41368 by Punnonen et al. “Optimization ofImmunomodulatory Properties of Genetic Vaccines;” EP 752008 by Stemmerand Crameri, “DNA Mutagenesis by Random Fragmentation and Reassembly;”EP 0932670 by Stemmer “Evolving Cellular DNA Uptake by RecursiveSequence Recombination;” WO 99/23107 by Stemmer et al., “Modification ofVirus Tropism and Host Range by Viral Genome Shuffling;” WO 99/21979 byApt et al., “Human Papillomavirus Vectors;” WO 98/31837 by del Cardayreet al. “Evolution of Whole Cells and Organisms by Recursive SequenceRecombination;” WO 98/27230 by Patten and Stemmer, “Methods andCompositions for Polypeptide Engineering;” WO 98/27230 by Stemmer etal., “Methods for Optimization of Gene Therapy by Recursive SequenceShuffling and Selection,” WO 00/00632, “Methods for Generating HighlyDiverse Libraries,” WO 00/09679, “Methods for Obtaining in VitroRecombined Polynucleotide Sequence Banks and Resulting Sequences,” WO98/42832 by Arnold et al., “Recombination of Polynucleotide SequencesUsing Random or Defined Primers,” WO 99/29902 by Arnold et al., “Methodfor Creating Polynucleotide and Polypeptide Sequences,” WO 98/41653 byVind, “An in Vitro Method for Construction of a DNA Library,” WO98/41622 by Borchert et al., “Method for Constructing a Library UsingDNA Shuffling,” and WO 98/42727 by Pati and Zarling, “SequenceAlterations using Homologous Recombination.”

Protocols that can be used to practice the invention (providing detailsregarding various diversity generating methods) are described, e.g., inU.S. patent application Ser. No. 09/407,800, “SHUFFLING OF CODON ALTEREDGENES” by Patten et al. filed Sep. 28, 1999; “EVOLUTION OF WHOLE CELLSAND ORGANISMS BY RECURSIVE SEQUENCE RECOMBINATION” by del Cardayre etal., U.S. Pat. No. 6,379,964; “OLIGONUCLEOTIDE MEDIATED NUCLEIC ACIDRECOMBINATION” by Crameri et al., U.S. Pat. Nos. 6,319,714; 6,368,861;6,376,246; 6,423,542; 6,426,224 and PCT/US00/01203; “USE OF CODON-VARIEDOLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING” by Welch et al., U.S.Pat. No. 6,436,675; “METHODS FOR MAKING CHARACTER STRINGS,POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS” bySelifonov et al., filed Jan. 18, 2000, (PCT/US00/01202) and, e.g.“METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDESHAVING DESIRED CHARACTERISTICS” by Selifonov et al., filed Jul. 18, 2000(U.S. Ser. No. 09/618,579); “METHODS OF POPULATING DATA STRUCTURES FORUSE IN EVOLUTIONARY SIMULATIONS” by Selifonov and Stemmer, filed Jan.18, 2000 (PCT/US00/01138); and “SINGLE-STRANDED NUCLEIC ACIDTEMPLATE-MEDIATED RECOMBINATION AND NUCLEIC ACID FRAGMENT ISOLATION” byAffholter, filed Sep. 6, 2000 (U.S. Ser. No. 09/656,549); and U.S. Pat.Nos. 6,177,263; 6,153,410.

Non-stochastic, or “directed evolution,” methods include, e.g.,saturation mutagenesis (GSSM), synthetic ligation reassembly (SLR), or acombination thereof are used to modify the nucleic acids of theinvention to generate amylases with new or altered properties (e.g.,activity under highly acidic or alkaline conditions, high temperatures,and the like). Polypeptides encoded by the modified nucleic acids can bescreened for an activity before testing for proteolytic or otheractivity. Any testing modality or protocol can be used, e.g., using acapillary array platform. See, e.g., U.S. Pat. Nos. 6,361,974;6,280,926; 5,939,250.

Saturation Mutagenesis, or, GSSM

In one aspect, codon primers containing a degenerate N,N,G/T sequenceare used to introduce point mutations into a polynucleotide, e.g., anamylase or an antibody of the invention, so as to generate a set ofprogeny polypeptides in which a full range of single amino acidsubstitutions is represented at each amino acid position, e.g., an aminoacid residue in an enzyme active site or ligand binding site targeted tobe modified. These oligonucleotides can comprise a contiguous firsthomologous sequence, a degenerate N,N,G/T sequence, and, optionally, asecond homologous sequence. The downstream progeny translationalproducts from the use of such oligonucleotides include all possibleamino acid changes at each amino acid site along the polypeptide,because the degeneracy of the N,N,G/T sequence includes codons for all20 amino acids. In one aspect, one such degenerate oligonucleotide(comprised of, e.g., one degenerate N,N,G/T cassette) is used forsubjecting each original codon in a parental polynucleotide template toa full range of codon substitutions. In another aspect, at least twodegenerate cassettes are used—either in the same oligonucleotide or not,for subjecting at least two original codons in a parental polynucleotidetemplate to a full range of codon substitutions. For example, more thanone N,N,G/T sequence can be contained in one oligonucleotide tointroduce amino acid mutations at more than one site. This plurality ofN,N,G/T sequences can be directly contiguous, or separated by one ormore additional nucleotide sequence(s). In another aspect,oligonucleotides serviceable for introducing additions and deletions canbe used either alone or in combination with the codons containing anN,N,G/T sequence, to introduce any combination or permutation of aminoacid additions, deletions, and/or substitutions.

In one aspect, simultaneous mutagenesis of two or more contiguous aminoacid positions is done using an oligonucleotide that contains contiguousN,N,G/T triplets, i.e. a degenerate (N,N,G/T)n sequence. In anotheraspect, degenerate cassettes having less degeneracy than the N,N,G/Tsequence are used. For example, it may be desirable in some instances touse (e.g. in an oligonucleotide) a degenerate triplet sequence comprisedof only one N, where said N can be in the first second or third positionof the triplet. Any other bases including any combinations andpermutations thereof can be used in the remaining two positions of thetriplet. Alternatively, it may be desirable in some instances to use(e.g. in an oligo) a degenerate N,N,N triplet sequence.

In one aspect, use of degenerate triplets (e.g., N,N,G/T triplets)allows for systematic and easy generation of a full range of possiblenatural amino acids (for a total of 20 amino acids) into each and everyamino acid position in a polypeptide (in alternative aspects, themethods also include generation of less than all possible substitutionsper amino acid residue, or codon, position). For example, for a 100amino acid polypeptide, 2000 distinct species (i.e. 20 possible aminoacids per position X 100 amino acid positions) can be generated. Throughthe use of an oligonucleotide or set of oligonucleotides containing adegenerate N,N,G/T triplet, 32 individual sequences can code for all 20possible natural amino acids. Thus, in a reaction vessel in which aparental polynucleotide sequence is subjected to saturation mutagenesisusing at least one such oligonucleotide, there are generated 32 distinctprogeny polynucleotides encoding 20 distinct polypeptides. In contrast,the use of a non-degenerate oligonucleotide in site-directed mutagenesisleads to only one progeny polypeptide product per reaction vessel.Nondegenerate oligonucleotides can optionally be used in combinationwith degenerate primers disclosed; for example, nondegenerateoligonucleotides can be used to generate specific point mutations in aworking polynucleotide. This provides one means to generate specificsilent point mutations, point mutations leading to corresponding aminoacid changes, and point mutations that cause the generation of stopcodons and the corresponding expression of polypeptide fragments.

In one aspect, each saturation mutagenesis reaction vessel containspolynucleotides encoding at least 20 progeny polypeptide (e.g.,amylases) molecules such that all 20 natural amino acids are representedat the one specific amino acid position corresponding to the codonposition mutagenized in the parental polynucleotide (other aspects useless than all 20 natural combinations). The 32-fold degenerate progenypolypeptides generated from each saturation mutagenesis reaction vesselcan be subjected to clonal amplification (e.g. cloned into a suitablehost, e.g., E. coli host, using, e.g., an expression vector) andsubjected to expression screening. When an individual progenypolypeptide is identified by screening to display a favorable change inproperty (when compared to the parental polypeptide, such as increasedproteolytic activity under alkaline or acidic conditions), it can besequenced to identify the correspondingly favorable amino acidsubstitution contained therein.

In one aspect, upon mutagenizing each and every amino acid position in aparental polypeptide using saturation mutagenesis as disclosed herein,favorable amino acid changes may be identified at more than one aminoacid position. One or more new progeny molecules can be generated thatcontain a combination of all or part of these favorable amino acidsubstitutions. For example, if 2 specific favorable amino acid changesare identified in each of 3 amino acid positions in a polypeptide, thepermutations include 3 possibilities at each position (no change fromthe original amino acid, and each of two favorable changes) and 3positions. Thus, there are 3×3×3 or 27 total possibilities, including 7that were previously examined—6 single point mutations (i.e. 2 at eachof three positions) and no change at any position.

In another aspect, site-saturation mutagenesis can be used together withanother stochastic or non-stochastic means to vary sequence, e.g.,synthetic ligation reassembly (see below), shuffling, chimerization,recombination and other mutagenizing processes and mutagenizing agents.This invention provides for the use of any mutagenizing process(es),including saturation mutagenesis, in an iterative manner.

Synthetic Ligation Reassembly (SLR)

The invention provides a non-stochastic gene modification system termed“synthetic ligation reassembly,” or simply “SLR,” a “directed evolutionprocess,” to generate polypeptides, e.g., amylases or antibodies of theinvention, with new or altered properties. SLR is a method of ligatingoligonucleotide fragments together non-stochastically. This methoddiffers from stochastic oligonucleotide shuffling in that the nucleicacid building blocks are not shuffled, concatenated or chimerizedrandomly, but rather are assembled non-stochastically. See, e.g., U.S.patent application Ser. No. 09/332,835 entitled “Synthetic LigationReassembly in Directed Evolution” and filed on Jun. 14, 1999 (“U.S. Ser.No. 09/332,835”). In one aspect, SLR comprises the following steps: (a)providing a template polynucleotide, wherein the template polynucleotidecomprises sequence encoding a homologous gene; (b) providing a pluralityof building block polynucleotides, wherein the building blockpolynucleotides are designed to cross-over reassemble with the templatepolynucleotide at a predetermined sequence, and a building blockpolynucleotide comprises a sequence that is a variant of the homologousgene and a sequence homologous to the template polynucleotide flankingthe variant sequence; (c) combining a building block polynucleotide witha template polynucleotide such that the building block polynucleotidecross-over reassembles with the template polynucleotide to generatepolynucleotides comprising homologous gene sequence variations.

SLR does not depend on the presence of high levels of homology betweenpolynucleotides to be rearranged. Thus, this method can be used tonon-stochastically generate libraries (or sets) of progeny moleculescomprised of over 10100 different chimeras. SLR can be used to generatelibraries comprised of over 101000 different progeny chimeras. Thus,aspects of the present invention include non-stochastic methods ofproducing a set of finalized chimeric nucleic acid molecule shaving anoverall assembly order that is chosen by design. This method includesthe steps of generating by design a plurality of specific nucleic acidbuilding blocks having serviceable mutually compatible ligatable ends,and assembling these nucleic acid building blocks, such that a designedoverall assembly order is achieved.

The mutually compatible ligatable ends of the nucleic acid buildingblocks to be assembled are considered to be “serviceable” for this typeof ordered assembly if they enable the building blocks to be coupled inpredetermined orders. Thus, the overall assembly order in which thenucleic acid building blocks can be coupled is specified by the designof the ligatable ends. If more than one assembly step is to be used,then the overall assembly order in which the nucleic acid buildingblocks can be coupled is also specified by the sequential order of theassembly step(s). In one aspect, the annealed building pieces aretreated with an enzyme, such as a ligase (e.g. T4 DNA ligase), toachieve covalent bonding of the building pieces.

In one aspect, the design of the oligonucleotide building blocks isobtained by analyzing a set of progenitor nucleic acid sequencetemplates that serve as a basis for producing a progeny set of finalizedchimeric polynucleotides. These parental oligonucleotide templates thusserve as a source of sequence information that aids in the design of thenucleic acid building blocks that are to be mutagenized, e.g.,chimerized or shuffled. In one aspect of this method, the sequences of aplurality of parental nucleic acid templates are aligned in order toselect one or more demarcation points. The demarcation points can belocated at an area of homology, and are comprised of one or morenucleotides. These demarcation points are preferably shared by at leasttwo of the progenitor templates. The demarcation points can thereby beused to delineate the boundaries of oligonucleotide building blocks tobe generated in order to rearrange the parental polynucleotides. Thedemarcation points identified and selected in the progenitor moleculesserve as potential chimerization points in the assembly of the finalchimeric progeny molecules. A demarcation point can be an area ofhomology (comprised of at least one homologous nucleotide base) sharedby at least two parental polynucleotide sequences. Alternatively, ademarcation point can be an area of homology that is shared by at leasthalf of the parental polynucleotide sequences, or, it can be an area ofhomology that is shared by at least two thirds of the parentalpolynucleotide sequences. Even more preferably a serviceable demarcationpoints is an area of homology that is shared by at least three fourthsof the parental polynucleotide sequences, or, it can be shared by atalmost all of the parental polynucleotide sequences. In one aspect, ademarcation point is an area of homology that is shared by all of theparental polynucleotide sequences.

In one aspect, a ligation reassembly process is performed exhaustivelyin order to generate an exhaustive library of progeny chimericpolynucleotides. In other words, all possible ordered combinations ofthe nucleic acid building blocks are represented in the set of finalizedchimeric nucleic acid molecules. At the same time, in another aspect,the assembly order (i.e. the order of assembly of each building block inthe 5′ to 3 sequence of each finalized chimeric nucleic acid) in eachcombination is by design (or non-stochastic) as described above. Becauseof the non-stochastic nature of this invention, the possibility ofunwanted side products is greatly reduced.

In another aspect, the ligation reassembly method is performedsystematically. For example, the method is performed in order togenerate a systematically compartmentalized library of progenymolecules, with compartments that can be screened systematically, e.g.one by one. In other words this invention provides that, through theselective and judicious use of specific nucleic acid building blocks,coupled with the selective and judicious use of sequentially steppedassembly reactions, a design can be achieved where specific sets ofprogeny products are made in each of several reaction vessels. Thisallows a systematic examination and screening procedure to be performed.Thus, these methods allow a potentially very large number of progenymolecules to be examined systematically in smaller groups. Because ofits ability to perform chimerizations in a manner that is highlyflexible yet exhaustive and systematic as well, particularly when thereis a low level of homology among the progenitor molecules, these methodsprovide for the generation of a library (or set) comprised of a largenumber of progeny molecules. Because of the non-stochastic nature of theinstant ligation reassembly invention, the progeny molecules generatedpreferably comprise a library of finalized chimeric nucleic acidmolecules having an overall assembly order that is chosen by design. Thesaturation mutagenesis and optimized directed evolution methods also canbe used to generate different progeny molecular species. It isappreciated that the invention provides freedom of choice and controlregarding the selection of demarcation points, the size and number ofthe nucleic acid building blocks, and the size and design of thecouplings. It is appreciated, furthermore, that the requirement forintermolecular homology is highly relaxed for the operability of thisinvention. In fact, demarcation points can even be chosen in areas oflittle or no intermolecular homology. For example, because of codonwobble, i.e. the degeneracy of codons, nucleotide substitutions can beintroduced into nucleic acid building blocks without altering the aminoacid originally encoded in the corresponding progenitor template.Alternatively, a codon can be altered such that the coding for anoriginally amino acid is altered. This invention provides that suchsubstitutions can be introduced into the nucleic acid building block inorder to increase the incidence of intermolecular homologous demarcationpoints and thus to allow an increased number of couplings to be achievedamong the building blocks, which in turn allows a greater number ofprogeny chimeric molecules to be generated.

In another aspect, the synthetic nature of the step in which thebuilding blocks are generated allows the design and introduction ofnucleotides (e.g., one or more nucleotides, which may be, for example,codons or introns or regulatory sequences) that can later be optionallyremoved in an in vitro process (e.g. by mutagenesis) or in an in vivoprocess (e.g. by utilizing the gene splicing ability of a hostorganism). It is appreciated that in many instances the introduction ofthese nucleotides may also be desirable for many other reasons inaddition to the potential benefit of creating a serviceable demarcationpoint.

In one aspect, a nucleic acid building block is used to introduce anintron. Thus, functional introns are introduced into a man-made genemanufactured according to the methods described herein. The artificiallyintroduced intron(s) can be functional in a host cells for gene splicingmuch in the way that naturally-occurring introns serve functionally ingene splicing.

Optimized Directed Evolution System

The invention provides a non-stochastic gene modification system termed“optimized directed evolution system” to generate polypeptides, e.g.,amylases or antibodies of the invention, with new or altered properties.Optimized directed evolution is directed to the use of repeated cyclesof reductive reassortment, recombination and selection that allow forthe directed molecular evolution of nucleic acids through recombination.Optimized directed evolution allows generation of a large population ofevolved chimeric sequences, wherein the generated population issignificantly enriched for sequences that have a predetermined number ofcrossover events.

A crossover event is a point in a chimeric sequence where a shift insequence occurs from one parental variant to another parental variant.Such a point is normally at the juncture of where oligonucleotides fromtwo parents are ligated together to form a single sequence. This methodallows calculation of the correct concentrations of oligonucleotidesequences so that the final chimeric population of sequences is enrichedfor the chosen number of crossover events. This provides more controlover choosing chimeric variants having a predetermined number ofcrossover events.

In addition, this method provides a convenient means for exploring atremendous amount of the possible protein variant space in comparison toother systems. Previously, if one generated, for example, 1013 chimericmolecules during a reaction, it would be extremely difficult to testsuch a high number of chimeric variants for a particular activity.Moreover, a significant portion of the progeny population would have avery high number of crossover events which resulted in proteins thatwere less likely to have increased levels of a particular activity. Byusing these methods, the population of chimerics molecules can beenriched for those variants that have a particular number of crossoverevents. Thus, although one can still generate 1013 chimeric moleculesduring a reaction, each of the molecules chosen for further analysismost likely has, for example, only three crossover events. Because theresulting progeny population can be skewed to have a predeterminednumber of crossover events, the boundaries on the functional varietybetween the chimeric molecules is reduced. This provides a moremanageable number of variables when calculating which oligonucleotidefrom the original parental polynucleotides might be responsible foraffecting a particular trait.

One method for creating a chimeric progeny polynucleotide sequence is tocreate oligonucleotides corresponding to fragments or portions of eachparental sequence. Each oligonucleotide preferably includes a uniqueregion of overlap so that mixing the oligonucleotides together resultsin a new variant that has each oligonucleotide fragment assembled in thecorrect order. Additional information can also be found, e.g., in U.S.Ser. No. 09/332,835; U.S. Pat. No. 6,361,974. The number ofoligonucleotides generated for each parental variant bears arelationship to the total number of resulting crossovers in the chimericmolecule that is ultimately created. For example, three parentalnucleotide sequence variants might be provided to undergo a ligationreaction in order to find a chimeric variant having, for example,greater activity at high temperature. As one example, a set of 50oligonucleotide sequences can be generated corresponding to eachportions of each parental variant. Accordingly, during the ligationreassembly process there could be up to 50 crossover events within eachof the chimeric sequences. The probability that each of the generatedchimeric polynucleotides will contain oligonucleotides from eachparental variant in alternating order is very low. If eacholigonucleotide fragment is present in the ligation reaction in the samemolar quantity it is likely that in some positions oligonucleotides fromthe same parental polynucleotide will ligate next to one another andthus not result in a crossover event. If the concentration of eacholigonucleotide from each parent is kept constant during any ligationstep in this example, there is a 1/3 chance (assuming 3 parents) that anoligonucleotide from the same parental variant will ligate within thechimeric sequence and produce no crossover.

Accordingly, a probability density function (PDF) can be determined topredict the population of crossover events that are likely to occurduring each step in a ligation reaction given a set number of parentalvariants, a number of oligonucleotides corresponding to each variant,and the concentrations of each variant during each step in the ligationreaction. The statistics and mathematics behind determining the PDF isdescribed below. By utilizing these methods, one can calculate such aprobability density function, and thus enrich the chimeric progenypopulation for a predetermined number of crossover events resulting froma particular ligation reaction. Moreover, a target number of crossoverevents can be predetermined, and the system then programmed to calculatethe starting quantities of each parental oligonucleotide during eachstep in the ligation reaction to result in a probability densityfunction that centers on the predetermined number of crossover events.These methods are directed to the use of repeated cycles of reductivereassortment, recombination and selection that allow for the directedmolecular evolution of a nucleic acid encoding a polypeptide throughrecombination. This system allows generation of a large population ofevolved chimeric sequences, wherein the generated population issignificantly enriched for sequences that have a predetermined number ofcrossover events. A crossover event is a point in a chimeric sequencewhere a shift in sequence occurs from one parental variant to anotherparental variant. Such a point is normally at the juncture of whereoligonucleotides from two parents are ligated together to form a singlesequence. The method allows calculation of the correct concentrations ofoligonucleotide sequences so that the final chimeric population ofsequences is enriched for the chosen number of crossover events. Thisprovides more control over choosing chimeric variants having apredetermined number of crossover events.

In addition, these methods provide a convenient means for exploring atremendous amount of the possible protein variant space in comparison toother systems. By using the methods described herein, the population ofchimerics molecules can be enriched for those variants that have aparticular number of crossover events. Thus, although one can stillgenerate 1013 chimeric molecules during a reaction, each of themolecules chosen for further analysis most likely has, for example, onlythree crossover events. Because the resulting progeny population can beskewed to have a predetermined number of crossover events, theboundaries on the functional variety between the chimeric molecules isreduced. This provides a more manageable number of variables whencalculating which oligonucleotide from the original parentalpolynucleotides might be responsible for affecting a particular trait.

In one aspect, the method creates a chimeric progeny polynucleotidesequence by creating oligonucleotides corresponding to fragments orportions of each parental sequence. Each oligonucleotide preferablyincludes a unique region of overlap so that mixing the oligonucleotidestogether results in a new variant that has each oligonucleotide fragmentassembled in the correct order. See also U.S. Ser. No. 09/332,835.

The number of oligonucleotides generated for each parental variant bearsa relationship to the total number of resulting crossovers in thechimeric molecule that is ultimately created. For example, threeparental nucleotide sequence variants might be provided to undergo aligation reaction in order to find a chimeric variant having, forexample, greater activity at high temperature. As one example, a set of50 oligonucleotide sequences can be generated corresponding to eachportions of each parental variant. Accordingly, during the ligationreassembly process there could be up to 50 crossover events within eachof the chimeric sequences. The probability that each of the generatedchimeric polynucleotides will contain oligonucleotides from eachparental variant in alternating order is very low. If eacholigonucleotide fragment is present in the ligation reaction in the samemolar quantity it is likely that in some positions oligonucleotides fromthe same parental polynucleotide will ligate next to one another andthus not result in a crossover event. If the concentration of eacholigonucleotide from each parent is kept constant during any ligationstep in this example, there is a 1/3 chance (assuming 3 parents) that anoligonucleotide from the same parental variant will ligate within thechimeric sequence and produce no crossover.

Accordingly, a probability density function (PDF) can be determined topredict the population of crossover events that are likely to occurduring each step in a ligation reaction given a set number of parentalvariants, a number of oligonucleotides corresponding to each variant,and the concentrations of each variant during each step in the ligationreaction. The statistics and mathematics behind determining the PDF isdescribed below. One can calculate such a probability density function,and thus enrich the chimeric progeny population for a predeterminednumber of crossover events resulting from a particular ligationreaction. Moreover, a target number of crossover events can bepredetermined, and the system then programmed to calculate the startingquantities of each parental oligonucleotide during each step in theligation reaction to result in a probability density function thatcenters on the predetermined number of crossover events.

Determining Crossover Events

Aspects of the invention include a system and software that receive adesired crossover probability density function (PDF), the number ofparent genes to be reassembled, and the number of fragments in thereassembly as inputs. The output of this program is a “fragment PDF”that can be used to determine a recipe for producing reassembled genes,and the estimated crossover PDF of those genes. The processing describedherein is preferably performed in MATLABâ (The Mathworks, Natick, Mass.)a programming language and development environment for technicalcomputing.

Iterative Processes

In practicing the invention, these processes can be iterativelyrepeated. For example, a nucleic acid (or, the nucleic acid) responsiblefor an altered or new amylase phenotype is identified, re-isolated,again modified, re-tested for activity. This process can be iterativelyrepeated until a desired phenotype is engineered. For example, an entirebiochemical anabolic or catabolic pathway can be engineered into a cell,including, e.g., starch hydrolysis activity.

Similarly, if it is determined that a particular oligonucleotide has noaffect at all on the desired trait (e.g., a new amylase phenotype), itcan be removed as a variable by synthesizing larger parentaloligonucleotides that include the sequence to be removed. Sinceincorporating the sequence within a larger sequence prevents anycrossover events, there will no longer be any variation of this sequencein the progeny polynucleotides. This iterative practice of determiningwhich oligonucleotides are most related to the desired trait, and whichare unrelated, allows more efficient exploration all of the possibleprotein variants that might be provide a particular trait or activity.

In Vivo Shuffling

In vivo shuffling of molecules is use in methods of the invention thatprovide variants of polypeptides of the invention, e.g., antibodies,amylases, and the like. In vivo shuffling can be performed utilizing thenatural property of cells to recombine multimers. While recombination invivo has provided the major natural route to molecular diversity,genetic recombination remains a relatively complex process thatinvolves 1) the recognition of homologies; 2) strand cleavage, strandinvasion, and metabolic steps leading to the production of recombinantchiasma; and finally 3) the resolution of chiasma into discreterecombined molecules. The formation of the chiasma requires therecognition of homologous sequences.

In one aspect, the invention provides a method for producing a hybridpolynucleotide from at least a first polynucleotide (e.g., an amylase ofthe invention) and a second polynucleotide (e.g., an enzyme, such as anamylase of the invention or any other amylase, or, a tag or an epitope).The invention can be used to produce a hybrid polynucleotide byintroducing at least a first polynucleotide and a second polynucleotidewhich share at least one region of partial sequence homology into asuitable host cell. The regions of partial sequence homology promoteprocesses which result in sequence reorganization producing a hybridpolynucleotide. The term “hybrid polynucleotide”, as used herein, is anynucleotide sequence which results from the method of the presentinvention and contains sequence from at least two originalpolynucleotide sequences. Such hybrid polynucleotides can result fromintermolecular recombination events which promote sequence integrationbetween DNA molecules. In addition, such hybrid polynucleotides canresult from intramolecular reductive reassortment processes whichutilize repeated sequences to alter a nucleotide sequence within a DNAmolecule.

Producing Sequence Variants

The invention also provides additional methods for making sequencevariants of the nucleic acid (e.g., amylase) sequences of the invention.The invention also provides additional methods for isolating amylasesusing the nucleic acids and polypeptides of the invention. In oneaspect, the invention provides for variants of an amylase codingsequence (e.g., a gene, cDNA or message) of the invention, which can bealtered by any means, including, e.g., random or stochastic methods, or,non-stochastic, or “directed evolution,” methods, as described above.

The isolated variants may be naturally occurring. Variant can also becreated in vitro. Variants may be created using genetic engineeringtechniques such as site directed mutagenesis, random chemicalmutagenesis, Exonuclease III deletion procedures, and standard cloningtechniques. Alternatively, such variants, fragments, analogs, orderivatives may be created using chemical synthesis or modificationprocedures. Other methods of making variants are also familiar to thoseskilled in the art. These include procedures in which nucleic acidsequences obtained from natural isolates are modified to generatenucleic acids which encode polypeptides having characteristics whichenhance their value in industrial or laboratory applications. In suchprocedures, a large number of variant sequences having one or morenucleotide differences with respect to the sequence obtained from thenatural isolate are generated and characterized. These nucleotidedifferences can result in amino acid changes with respect to thepolypeptides encoded by the nucleic acids from the natural isolates.

For example, variants may be created using error prone PCR. In errorprone PCR, PCR is performed under conditions where the copying fidelityof the DNA polymerase is low, such that a high rate of point mutationsis obtained along the entire length of the PCR product. Error prone PCRis described, e.g., in Leung, D. W., et al., Technique, 1:11-15, 1989)and Caldwell, R. C. & Joyce G. F., PCR Methods Applic., 2:28-33, 1992.Briefly, in such procedures, nucleic acids to be mutagenized are mixedwith PCR primers, reaction buffer, MgCl₂, MnCl₂, Taq polymerase and anappropriate concentration of dNTPs for achieving a high rate of pointmutation along the entire length of the PCR product. For example, thereaction may be performed using 20 fmoles of nucleic acid to bemutagenized, 30 pmole of each PCR primer, a reaction buffer comprising50 mM KCl, 10 mM Tris HCl (pH 8.3) and 0.01% gelatin, 7 mM MgCl₂, 0.5 mMMnCl₂, 5 units of Taq polymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP,and 1 mM dTTP. PCR may be performed for 30 cycles of 94° C. for 1 min,45° C. for 1 min, and 72° C. for 1 min. However, it will be appreciatedthat these parameters may be varied as appropriate. The mutagenizednucleic acids are cloned into an appropriate vector and the activitiesof the polypeptides encoded by the mutagenized nucleic acids isevaluated.

Variants may also be created using oligonucleotide directed mutagenesisto generate site-specific mutations in any cloned DNA of interest.Oligonucleotide mutagenesis is described, e.g., in Reidhaar-Olson (1988)Science 241:53-57. Briefly, in such procedures a plurality of doublestranded oligonucleotides bearing one or more mutations to be introducedinto the cloned DNA are synthesized and inserted into the cloned DNA tobe mutagenized. Clones containing the mutagenized DNA are recovered andthe activities of the polypeptides they encode are assessed.

Another method for generating variants is assembly PCR. Assembly PCRinvolves the assembly of a PCR product from a mixture of small DNAfragments. A large number of different PCR reactions occur in parallelin the same vial, with the products of one reaction priming the productsof another reaction. Assembly PCR is described in, e.g., U.S. Pat. No.5,965,408.

Still another method of generating variants is sexual PCR mutagenesis.In sexual PCR mutagenesis, forced homologous recombination occursbetween DNA molecules of different but highly related DNA sequence invitro, as a result of random fragmentation of the DNA molecule based onsequence homology, followed by fixation of the crossover by primerextension in a PCR reaction. Sexual PCR mutagenesis is described, e.g.,in Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751. Briefly, insuch procedures a plurality of nucleic acids to be recombined aredigested with DNase to generate fragments having an average size of50-200 nucleotides. Fragments of the desired average size are purifiedand resuspended in a PCR mixture. PCR is conducted under conditionswhich facilitate recombination between the nucleic acid fragments. Forexample, PCR may be performed by resuspending the purified fragments ata concentration of 10-30 ng/:1 in a solution of 0.2 mM of each dNTP, 2.2mM MgCl₂, 50 mM KCL, 10 mM Tris HCl, pH 9.0, and 0.1% Triton X-100. 2.5units of Taq polymerase per 100:1 of reaction mixture is added and PCRis performed using the following regime: 94° C. for 60 seconds, 94° C.for 30 seconds, 50-55° C. for 30 seconds, 72° C. for 30 seconds (30-45times) and 72° C. for 5 minutes. However, it will be appreciated thatthese parameters may be varied as appropriate. In some aspects,oligonucleotides may be included in the PCR reactions. In other aspects,the Klenow fragment of DNA polymerase I may be used in a first set ofPCR reactions and Taq polymerase may be used in a subsequent set of PCRreactions. Recombinant sequences are isolated and the activities of thepolypeptides they encode are assessed.

Variants may also be created by in vivo mutagenesis. In some aspects,random mutations in a sequence of interest are generated by propagatingthe sequence of interest in a bacterial strain, such as an E. colistrain, which carries mutations in one or more of the DNA repairpathways. Such “mutator” strains have a higher random mutation rate thanthat of a wild-type parent. Propagating the DNA in one of these strainswill eventually generate random mutations within the DNA. Mutatorstrains suitable for use for in vivo mutagenesis are described, e.g., inPCT Publication No. WO 91/16427.

Variants may also be generated using cassette mutagenesis. In cassettemutagenesis a small region of a double stranded DNA molecule is replacedwith a synthetic oligonucleotide “cassette” that differs from the nativesequence. The oligonucleotide often contains completely and/or partiallyrandomized native sequence.

Recursive ensemble mutagenesis may also be used to generate variants.Recursive ensemble mutagenesis is an algorithm for protein engineering(protein mutagenesis) developed to produce diverse populations ofphenotypically related mutants whose members differ in amino acidsequence. This method uses a feedback mechanism to control successiverounds of combinatorial cassette mutagenesis. Recursive ensemblemutagenesis is described, e.g., in Arkin (1992) Proc. Natl. Acad. Sci.USA 89:7811-7815.

In some aspects, variants are created using exponential ensemblemutagenesis. Exponential ensemble mutagenesis is a process forgenerating combinatorial libraries with a high percentage of unique andfunctional mutants, wherein small groups of residues are randomized inparallel to identify, at each altered position, amino acids which leadto functional proteins. Exponential ensemble mutagenesis is described,e.g., in Delegrave (1993) Biotechnology Res. 11:1548-1552. Random andsite-directed mutagenesis are described, e.g., in Arnold (1993) CurrentOpinion in Biotechnology 4:450-455.

In some aspects, the variants are created using shuffling procedureswherein portions of a plurality of nucleic acids which encode distinctpolypeptides are fused together to create chimeric nucleic acidsequences which encode chimeric polypeptides as described in, e.g., U.S.Pat. Nos. 5,965,408; 5,939,250 (see also discussion, above).

The invention also provides variants of polypeptides of the invention(e.g., amylases) comprising sequences in which one or more of the aminoacid residues (e.g., of an exemplary polypeptide, such as SEQ ID NO:2;SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO:10; SEQ ID NO:12) aresubstituted with a conserved or non-conserved amino acid residue (e.g.,a conserved amino acid residue) and such substituted amino acid residuemay or may not be one encoded by the genetic code. Conservativesubstitutions are those that substitute a given amino acid in apolypeptide by another amino acid of like characteristics. Thus,polypeptides of the invention include those with conservativesubstitutions of sequences of the invention, e.g., the exemplary SEQ IDNO:2; SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO:10; SEQ ID NO:12,including but not limited to the following replacements: replacements ofan aliphatic amino acid such as Alanine, Valine, Leucine and Isoleucinewith another aliphatic amino acid; replacement of a Serine with aThreonine or vice versa; replacement of an acidic residue such asAspartic acid and Glutamic acid with another acidic residue; replacementof a residue bearing an amide group, such as Asparagine and Glutamine,with another residue bearing an amide group; exchange of a basic residuesuch as Lysine and Arginine with another basic residue; and replacementof an aromatic residue such as Phenylalanine, Tyrosine with anotheraromatic residue. Other variants are those in which one or more of theamino acid residues of the polypeptides of the invention includes asubstituent group.

Other variants within the scope of the invention are those in which thepolypeptide is associated with another compound, such as a compound toincrease the half-life of the polypeptide, for example, polyethyleneglycol.

Additional variants within the scope of the invention are those in whichadditional amino acids are fused to the polypeptide, such as a leadersequence, a secretory sequence, a proprotein sequence or a sequencewhich facilitates purification, enrichment, or stabilization of thepolypeptide.

In some aspects, the variants, fragments, derivatives and analogs of thepolypeptides of the invention retain the same biological function oractivity as the exemplary polypeptides, e.g., amylase activity, asdescribed herein. In other aspects, the variant, fragment, derivative,or analog includes a proprotein, such that the variant, fragment,derivative, or analog can be activated by cleavage of the proproteinportion to produce an active polypeptide.

Optimizing Codons to Achieve High Levels of Protein Expression in HostCells

The invention provides methods for modifying amylase-encoding nucleicacids to modify codon usage. In one aspect, the invention providesmethods for modifying codons in a nucleic acid encoding an amylase toincrease or decrease its expression in a host cell. The invention alsoprovides nucleic acids encoding an amylase modified to increase itsexpression in a host cell, amylase so modified, and methods of makingthe modified amylases. The method comprises identifying a“non-preferred” or a “less preferred” codon in amylase-encoding nucleicacid and replacing one or more of these non-preferred or less preferredcodons with a “preferred codon” encoding the same amino acid as thereplaced codon and at least one non-preferred or less preferred codon inthe nucleic acid has been replaced by a preferred codon encoding thesame amino acid. A preferred codon is a codon over-represented in codingsequences in genes in the host cell and a non-preferred or lesspreferred codon is a codon under-represented in coding sequences ingenes in the host cell.

Host cells for expressing the nucleic acids, expression cassettes andvectors of the invention include bacteria, yeast, fungi, plant cells,insect cells and mammalian cells. Thus, the invention provides methodsfor optimizing codon usage in all of these cells, codon-altered nucleicacids and polypeptides made by the codon-altered nucleic acids.Exemplary host cells include gram negative bacteria, such as Escherichiacoli and Pseudomonas fluorescens; gram positive bacteria, such asStreptomyces diversa, Lactobacillus gasseri, Lactococcus lactis,Lactococcus cremoris, Bacillus subtilis. Exemplary host cells alsoinclude eukaryotic organisms, e.g., various yeast, such as Saccharomycessp., including Saccharomyces cerevisiae, Schizosaccharomyces pombe,Pichia pastoris, and Kluyveromyces lactis, Hansenula polymorpha,Aspergillus niger, and mammalian cells and cell lines and insect cellsand cell lines. Thus, the invention also includes nucleic acids andpolypeptides optimized for expression in these organisms and species.

For example, the codons of a nucleic acid encoding an amylase isolatedfrom a bacterial cell are modified such that the nucleic acid isoptimally expressed in a bacterial cell different from the bacteria fromwhich the amylase was derived, a yeast, a fungi, a plant cell, an insectcell or a mammalian cell. Methods for optimizing codons are well knownin the art, see, e.g., U.S. Pat. No. 5,795,737; Baca (2000) Int. J.Parasitol. 30:113-118; Hale (1998) Protein Expr. Purif. 12:185-188;Narum (2001) Infect. Immun. 69:7250-7253. See also Narum (2001) Infect.Immun. 69:7250-7253, describing optimizing codons in mouse systems;Outchkourov (2002) Protein Expr. Purif. 24:18-24, describing optimizingcodons in yeast; Feng (2000) Biochemistry 39:15399-15409, describingoptimizing codons in E. coli; Humphreys (2000) Protein Expr. Purif.20:252-264, describing optimizing codon usage that affects secretion inE. coli.

Transgenic Non-Human Animals

The invention provides transgenic non-human animals comprising a nucleicacid, a polypeptide, an expression cassette or vector or a transfectedor transformed cell of the invention. The invention also providesmethods of making and using these transgenic non-human animals.

The transgenic non-human animals can be, e.g., goats, rabbits, sheep,pigs, cows, rats and mice, comprising the nucleic acids of theinvention. These animals can be used, e.g., as in vivo models to studyamylase activity, or, as models to screen for agents that change theamylase activity in vivo. The coding sequences for the polypeptides tobe expressed in the transgenic non-human animals can be designed to beconstitutive, or, under the control of tissue-specific,developmental-specific or inducible transcriptional regulatory factors.Transgenic non-human animals can be designed and generated using anymethod known in the art; see, e.g., U.S. Pat. Nos. 6,211,428; 6,187,992;6,156,952; 6,118,044; 6,111,166; 6,107,541; 5,959,171; 5,922,854;5,892,070; 5,880,327; 5,891,698; 5,639,940; 5,573,933; 5,387,742;5,087,571, describing making and using transformed cells and eggs andtransgenic mice, rats, rabbits, sheep, pigs and cows. See also, e.g.,Pollock (1999) J. Immunol. Methods 231:147-157, describing theproduction of recombinant proteins in the milk of transgenic dairyanimals; Baguisi (1999) Nat. Biotechnol. 17:456-461, demonstrating theproduction of transgenic goats. U.S. Pat. No. 6,211,428, describesmaking and using transgenic non-human mammals which express in theirbrains a nucleic acid construct comprising a DNA sequence. U.S. Pat. No.5,387,742, describes injecting cloned recombinant or synthetic DNAsequences into fertilized mouse eggs, implanting the injected eggs inpseudo-pregnant females, and growing to term transgenic mice whose cellsexpress proteins related to the pathology of Alzheimer's disease. U.S.Pat. No. 6,187,992, describes making and using a transgenic mouse whosegenome comprises a disruption of the gene encoding amyloid precursorprotein (APP).

“Knockout animals” can also be used to practice the methods of theinvention. For example, in one aspect, the transgenic or modifiedanimals of the invention comprise a “knockout animal,” e.g., a “knockoutmouse,” engineered not to express an endogenous gene, which is replacedwith a gene expressing an amylase of the invention, or, a fusion proteincomprising an amylase of the invention.

Transgenic Plants and Seeds

The invention provides transgenic plants and seeds comprising a nucleicacid, a polypeptide, an expression cassette or vector or a transfectedor transformed cell of the invention. The transgenic plant can bedicotyledonous (a dicot) or monocotyledonous (a monocot). The inventionalso provides methods of making and using these transgenic plants andseeds. The transgenic plant or plant cell expressing a polypeptide ofthe present invention may be constructed in accordance with any methodknown in the art. See, for example, U.S. Pat. No. 6,309,872.

Nucleic acids and expression constructs of the invention can beintroduced into a plant cell by any means. For example, nucleic acids orexpression constructs can be introduced into the genome of a desiredplant host, or, the nucleic acids or expression constructs can beepisomes. Introduction into the genome of a desired plant can be suchthat the host's α-amylase production is regulated by endogenoustranscriptional or translational control elements. The invention alsoprovides “knockout plants” where insertion of gene sequence by, e.g.,homologous recombination, has disrupted the expression of the endogenousgene. Means to generate “knockout” plants are well-known in the art,see, e.g., Strepp (1998) Proc Natl. Acad. Sci. USA 95:4368-4373; Miao(1995) Plant J 7:359-365. See discussion on transgenic plants, below.

The nucleic acids of the invention can be used to confer desired traitson essentially any plant, e.g., on starch-producing plants, such aspotato, wheat, rice, barley, and the like. Nucleic acids of theinvention can be used to manipulate metabolic pathways of a plant inorder to optimize or alter host's expression of α-amylase. The canchange the ratio of starch/sugar conversion in a plant. This canfacilitate industrial processing of a plant. Alternatively,alpha-amylases of the invention can be used in production of atransgenic plant to produce a compound not naturally produced by thatplant. This can lower production costs or create a novel product.

In one aspect, the first step in production of a transgenic plantinvolves making an expression construct for expression in a plant cell.These techniques are well known in the art. They can include selectingand cloning a promoter, a coding sequence for facilitating efficientbinding of ribosomes to mRNA and selecting the appropriate geneterminator sequences. One exemplary constitutive promoter is CaMV35S,from the cauliflower mosaic virus, which generally results in a highdegree of expression in plants. Other promoters are more specific andrespond to cues in the plant's internal or external environment. Anexemplary light-inducible promoter is the promoter from the cab gene,encoding the major chlorophyll a/b binding protein.

In one aspect, the nucleic acid is modified to achieve greaterexpression in a plant cell. For example, a sequence of the invention islikely to have a higher percentage of A-T nucleotide pairs compared tothat seen in a plant, some of which prefer G-C nucleotide pairs.Therefore, A-T nucleotides in the coding sequence can be substitutedwith G-C nucleotides without significantly changing the amino acidsequence to enhance production of the gene product in plant cells.

Selectable marker gene can be added to the gene construct in order toidentify plant cells or tissues that have successfully integrated thetransgene. This may be necessary because achieving incorporation andexpression of genes in plant cells is a rare event, occurring in just afew percent of the targeted tissues or cells. Selectable marker genesencode proteins that provide resistance to agents that are normallytoxic to plants, such as antibiotics or herbicides. Only plant cellsthat have integrated the selectable marker gene will survive when grownon a medium containing the appropriate antibiotic or herbicide. As forother inserted genes, marker genes also require promoter and terminationsequences for proper function.

In one aspect, making transgenic plants or seeds comprises incorporatingsequences of the invention and, optionally, marker genes into a targetexpression construct (e.g., a plasmid), along with positioning of thepromoter and the terminator sequences. This can involve transferring themodified gene into the plant through a suitable method. For example, aconstruct may be introduced directly into the genomic DNA of the plantcell using techniques such as electroporation and microinjection ofplant cell protoplasts, or the constructs can be introduced directly toplant tissue using ballistic methods, such as DNA particle bombardment.For example, see, e.g., Christou (1997) Plant Mol. Biol. 35:197-203;Pawlowski (1996) Mol. Biotechnol. 6:17-30; Klein (1987) Nature327:70-73; Takumi (1997) Genes Genet. Syst. 72:63-69, discussing use ofparticle bombardment to introduce transgenes into wheat; and Adam (1997)supra, for use of particle bombardment to introduce YACs into plantcells. For example, Rinehart (1997) supra, used particle bombardment togenerate transgenic cotton plants. Apparatus for accelerating particlesis described U.S. Pat. No. 5,015,580; and, the commercially availableBioRad (Biolistics) PDS-2000 particle acceleration instrument; see also,John, U.S. Pat. No. 5,608,148; and Ellis, U.S. Pat. No. 5,681,730,describing particle-mediated transformation of gymnosperms.

In one aspect, protoplasts can be immobilized and injected with anucleic acids, e.g., an expression construct. Although plantregeneration from protoplasts is not easy with cereals, plantregeneration is possible in legumes using somatic embryogenesis fromprotoplast derived callus. Organized tissues can be transformed withnaked DNA using gene gun technique, where DNA is coated on tungstenmicroprojectiles, shot 1/100th the size of cells, which carry the DNAdeep into cells and organelles. Transformed tissue is then induced toregenerate, usually by somatic embryogenesis. This technique has beensuccessful in several cereal species including maize and rice.

Nucleic acids, e.g., expression constructs, can also be introduced in toplant cells using recombinant viruses. Plant cells can be transformedusing viral vectors, such as, e.g., tobacco mosaic virus derived vectors(Rouwendal (1997) Plant Mol. Biol. 33:989-999), see Porta (1996) “Use ofviral replicons for the expression of genes in plants,” Mol. Biotechnol.5:209-221.

Alternatively, nucleic acids, e.g., an expression construct, can becombined with suitable T-DNA flanking regions and introduced into aconventional Agrobacterium tumefaciens host vector. The virulencefunctions of the Agrobacterium tumefaciens host will direct theinsertion of the construct and adjacent marker into the plant cell DNAwhen the cell is infected by the bacteria. Agrobacteriumtumefaciens-mediated transformation techniques, including disarming anduse of binary vectors, are well described in the scientific literature.See, e.g., Horsch (1984) Science 233:496-498; Fraley (1983) Proc. Natl.Acad. Sci. USA 80:4803 (1983); Gene Transfer to Plants, Potrykus, ed.(Springer-Verlag, Berlin 1995). The DNA in an A. tumefaciens cell iscontained in the bacterial chromosome as well as in another structureknown as a Ti (tumor-inducing) plasmid. The Ti plasmid contains astretch of DNA termed T-DNA (˜20 kb long) that is transferred to theplant cell in the infection process and a series of vir (virulence)genes that direct the infection process. A. tumefaciens can only infecta plant through wounds: when a plant root or stem is wounded it givesoff certain chemical signals, in response to which, the vir genes of A.tumefaciens become activated and direct a series of events necessary forthe transfer of the T-DNA from the Ti plasmid to the plant's chromosome.The T-DNA then enters the plant cell through the wound. One speculationis that the T-DNA waits until the plant DNA is being replicated ortranscribed, then inserts itself into the exposed plant DNA. In order touse A. tumefaciens as a transgene vector, the tumor-inducing section ofT-DNA have to be removed, while retaining the T-DNA border regions andthe vir genes. The transgene is then inserted between the T-DNA borderregions, where it is transferred to the plant cell and becomesintegrated into the plant's chromosomes.

The invention provides for the transformation of monocotyledonous plantsusing the nucleic acids of the invention, including important cereals,see Hiei (1997) Plant Mol. Biol. 35:205-218. See also, e.g., Horsch,Science (1984) 233:496; Fraley (1983) Proc. Natl. Acad. Sci. USA80:4803; Thykjaer (1997) supra; Park (1996) Plant Mol. Biol.32:1135-1148, discussing T-DNA integration into genomic DNA. See alsoD'Halluin, U.S. Pat. No. 5,712,135, describing a process for the stableintegration of a DNA comprising a gene that is functional in a cell of acereal, or other monocotyledonous plant.

In one aspect, the third step can involve selection and regeneration ofwhole plants capable of transmitting the incorporated target gene to thenext generation. Such regeneration techniques rely on manipulation ofcertain phytohormones in a tissue culture growth medium, typicallyrelying on a biocide and/or herbicide marker that has been introducedtogether with the desired nucleotide sequences. Plant regeneration fromcultured protoplasts is described in Evans et al., Protoplasts Isolationand Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilanPublishing Company, New York, 1983; and Binding, Regeneration of Plants,Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regenerationcan also be obtained from plant callus, explants, organs, or partsthereof. Such regeneration techniques are described generally in Klee(1987) Ann. Rev. of Plant Phys. 38:467-486. To obtain whole plants fromtransgenic tissues such as immature embryos, they can be grown undercontrolled environmental conditions in a series of media containingnutrients and hormones, a process known as tissue culture. Once wholeplants are generated and produce seed, evaluation of the progeny begins.

After the expression cassette is stably incorporated in transgenicplants, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed. Since transgenic expression of the nucleicacids of the invention leads to phenotypic changes, plants comprisingthe recombinant nucleic acids of the invention can be sexually crossedwith a second plant to obtain a final product. Thus, the seed of theinvention can be derived from a cross between two transgenic plants ofthe invention, or a cross between a plant of the invention and anotherplant. The desired effects (e.g., expression of the polypeptides of theinvention to produce a plant in which flowering behavior is altered) canbe enhanced when both parental plants express the polypeptides of theinvention. The desired effects can be passed to future plant generationsby standard propagation means.

The nucleic acids and polypeptides of the invention are expressed in orinserted in any plant or seed. Transgenic plants of the invention can bedicotyledonous or monocotyledonous. Examples of monocot transgenicplants of the invention are grasses, such as meadow grass (blue grass,Poa), forage grass such as festuca, lolium, temperate grass, such asAgrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum,and maize (corn). Examples of dicot transgenic plants of the inventionare tobacco, legumes, such as lupins, potato, sugar beet, pea, bean andsoybean, and cruciferous plants (family Brassicaceae), such ascauliflower, rape seed, and the closely related model organismArabidopsis thaliana. Thus, the transgenic plants and seeds of theinvention include a broad range of plants, including, but not limitedto, species from the genera Anacardium, Arachis, Asparagus, Atropa,Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea,Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium,Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium,Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana,Olea, Oryza, Panieum, Pannisetum, Persea, Phaseolus, Pistachia, Pisum,Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum,Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna, and Zea.

In alternative embodiments, the nucleic acids of the invention areexpressed in plants which contain fiber cells, including, e.g., cotton,silk cotton tree (Kapok, Ceiba pentandra), desert willow, creosote bush,winterfat, balsa, ramie, kenaf, hemp, roselle, jute, sisal abaca andflax. In alternative embodiments, the transgenic plants of the inventioncan be members of the genus Gossypium, including members of anyGossypium species, such as G. arboreum; G. herbaceum, G. barbadense, andG. hirsutum.

The invention also provides for transgenic plants to be used forproducing large amounts of the polypeptides of the invention. Forexample, see Palmgren (1997) Trends Genet. 13:348; Chong (1997)Transgenic Res. 6:289-296 (producing human milk protein beta-casein intransgenic potato plants using an auxin-inducible, bidirectionalmannopine synthase (mas 1′,2′) promoter with Agrobacteriumtumefaciens-mediated leaf disc transformation methods).

Using known procedures, one of skill can screen for plants of theinvention by detecting the increase or decrease of transgene mRNA orprotein in transgenic plants. Means for detecting and quantitation ofmRNAs or proteins are well known in the art.

Polypeptides and Peptides

The invention provides isolated or recombinant polypeptides having asequence identity (e.g., at least about 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more, or complete (100%) sequence identity) to an exemplarysequence of the invention, e.g., SEQ ID NO:2; SEQ ID NO:6; SEQ ID NO:8,SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16. As discussed above, theidentity can be over the full length of the polypeptide, or, theidentity can be over a region of at least about 50, 60, 70, 80, 90, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700 or moreresidues. Polypeptides of the invention can also be shorter than thefull length of exemplary polypeptides (e.g., SEQ ID NO:2; SEQ ID NO:6;SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16).In alternative aspects, the invention provides polypeptides (peptides,fragments) ranging in size between about 5 and the full length of apolypeptide, e.g., an enzyme, such as an amylase; exemplary sizes beingof about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, or more residues, e.g., contiguous residues of an exemplaryamylase of the invention. Peptides of the invention can be useful as,e.g., labeling probes, antigens, toleragens, motifs, amylase activesites.

The polypeptides of the invention include amylases in an active orinactive form. For example, the polypeptides of the invention includeproproteins before “maturation” or processing of prepro sequences, e.g.,by a proprotein-processing enzyme, such as a proprotein convertase togenerate an “active” mature protein. The polypeptides of the inventioninclude proteases inactive for other reasons, e.g., before “activation”by a post-translational processing event, e.g., an endo- orexo-peptidase or proteinase action, a phosphorylation event, anamidation, a glycosylation or a sulfation, a dimerization event, and thelike.

The polypeptides of the invention include all active forms, includingactive subsequences, e.g., catalytic domains or active sites, ofamylases of the invention. In one aspect, the invention providescatalytic domains or active sites as determined by a software paradigm,e.g., Pfam. In one aspect, the invention provides a peptide orpolypeptide comprising or consisting of an active site domain aspredicted through use of a database, e.g., Pfam, which is a largecollection of multiple sequence alignments and hidden Markov modelscovering many common protein families, The Pfam protein familiesdatabase, A. Bateman, E. Birney, L. Cerruti, R. Durbin, L. Etwiller, S.R. Eddy, S. Griffiths-Jones, K. L. Howe, M. Marshall, and E. L. L.Sonnhammer, Nucleic Acids Research, 30(1):276-280, 2002).

Methods for identifying “prepro” domain sequences and signal sequencesare well known in the art, see, e.g., Van de Ven (1993) Crit. Rev.Oncog. 4(2):115-136. For example, to identify a prepro sequence, theprotein is purified from the extracellular space and the N-terminalprotein sequence is determined and compared to the unprocessed form.

The invention includes polypeptides with or without a signal sequenceand/or a prepro sequence. The invention includes polypeptides withheterologous signal sequences and/or prepro sequences. The preprosequence (including a sequence of the invention used as a heterologousprepro domain) can be located on the amino terminal or the carboxyterminal end of the protein. The invention also includes isolated orrecombinant signal sequences, prepro sequences and catalytic domains(e.g., “active sites”) comprising sequences of the invention.

The percent sequence identity can be over the full length of thepolypeptide, or, the identity can be over a region of at least about 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100,125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700 ormore residues. Polypeptides of the invention can also be shorter thanthe full length of exemplary polypeptides. In alternative aspects, theinvention provides polypeptides (peptides, fragments) ranging in sizebetween about 5 and the full length of a polypeptide, e.g., an enzyme,such as a protease; exemplary sizes being of about 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or more residues,e.g., contiguous residues of an exemplary amylase of the invention.

Peptides of the invention (e.g., a subsequence of an exemplarypolypeptide of the invention) can be useful as, e.g., labeling probes,antigens, toleragens, motifs, amylase active sites (e.g., “catalyticdomains”), signal sequences and/or prepro domains.

Polypeptides and peptides of the invention can be isolated from naturalsources, be synthetic, or be recombinantly generated polypeptides.Peptides and proteins can be recombinantly expressed in vitro or invivo. The peptides and polypeptides of the invention can be made andisolated using any method known in the art. Polypeptide and peptides ofthe invention can also be synthesized, whole or in part, using chemicalmethods well known in the art. See e.g., Caruthers (1980) Nucleic AcidsRes. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res. Symp. Ser.225-232; Banga, A. K., Therapeutic Peptides and Proteins, Formulation,Processing and Delivery Systems (1995) Technomic Publishing Co.,Lancaster, Pa. For example, peptide synthesis can be performed usingvarious solid-phase techniques (see e.g., Roberge (1995) Science269:202; Merrifield (1997) Methods Enzymol. 289:3-13) and automatedsynthesis may be achieved, e.g., using the ABI 431A Peptide Synthesizer(Perkin Elmer) in accordance with the instructions provided by themanufacturer.

The peptides and polypeptides of the invention can also be glycosylated.The glycosylation can be added post-translationally either chemically orby cellular biosynthetic mechanisms, wherein the later incorporates theuse of known glycosylation motifs, which can be native to the sequenceor can be added as a peptide or added in the nucleic acid codingsequence. The glycosylation can be O-linked or N-linked.

The peptides and polypeptides of the invention, as defined above,include all “mimetic” and “peptidomimetic” forms. The terms “mimetic”and “peptidomimetic” refer to a synthetic chemical compound which hassubstantially the same structural and/or functional characteristics ofthe polypeptides of the invention. The mimetic can be either entirelycomposed of synthetic, non-natural analogues of amino acids, or, is achimeric molecule of partly natural peptide amino acids and partlynon-natural analogs of amino acids. The mimetic can also incorporate anyamount of natural amino acid conservative substitutions as long as suchsubstitutions also do not substantially alter the mimetic's structureand/or activity. As with polypeptides of the invention which areconservative variants, routine experimentation will determine whether amimetic is within the scope of the invention, i.e., that its structureand/or function is not substantially altered. Thus, in one aspect, amimetic composition is within the scope of the invention if it has anamylase activity.

Polypeptide mimetic compositions of the invention can contain anycombination of non-natural structural components. In alternative aspect,mimetic compositions of the invention include one or all of thefollowing three structural groups: a) residue linkage groups other thanthe natural amide bond (“peptide bond”) linkages; b) non-naturalresidues in place of naturally occurring amino acid residues; or c)residues which induce secondary structural mimicry, i.e., to induce orstabilize a secondary structure, e.g., a beta turn, gamma turn, betasheet, alpha helix conformation, and the like. For example, apolypeptide of the invention can be characterized as a mimetic when allor some of its residues are joined by chemical means other than naturalpeptide bonds. Individual peptidomimetic residues can be joined bypeptide bonds, other chemical bonds or coupling means, such as, e.g.,glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides,N,N′-dicyclohexylcarbodiimide (DCC) or N,N′-diisopropylcarbodiimide(DIC). Linking groups that can be an alternative to the traditionalamide bond (“peptide bond”) linkages include, e.g., ketomethylene (e.g.,—C(═O)—CH₂— for —C(═O)—NH—), aminomethylene (CH₂—NH), ethylene, olefin(CH═CH), ether (CH₂—O), thioether (CH₂—S), tetrazole (CN₄—), thiazole,retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistryand Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp267-357, “Peptide Backbone Modifications,” Marcell Dekker, N.Y.).

A polypeptide of the invention can also be characterized as a mimetic bycontaining all or some non-natural residues in place of naturallyoccurring amino acid residues. Non-natural residues are well describedin the scientific and patent literature; a few exemplary non-naturalcompositions useful as mimetics of natural amino acid residues andguidelines are described below. Mimetics of aromatic amino acids can begenerated by replacing by, e.g., D- or L-naphylalanine; D- orL-phenylglycine; D- or L-2 thienylalanine; D- or L-1, -2,3-, or4-pyrenylalanine; D- or L-3 thienylalanine; D- orL-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- orL-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine;D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine;D-p-fluoro-phenylalanine; D- or L-p-biphenylphenylalanine; K- orL-p-methoxy-biphenylphenylalanine; D- or L-2-indole(alkyl)alanines; and,D- or L-alkylainines, where alkyl can be substituted or unsubstitutedmethyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl,sec-isotyl, iso-pentyl, or a non-acidic amino acids. Aromatic rings of anon-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl,benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.

Mimetics of acidic amino acids can be generated by substitution by,e.g., non-carboxylate amino acids while maintaining a negative charge;(phosphono)alanine; sulfated threonine. Carboxyl side groups (e.g.,aspartyl or glutamyl) can also be selectively modified by reaction withcarbodiimides (R′—N—C—N—R′) such as, e.g.,1-cyclohexyl-3(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide. Aspartyl orglutamyl can also be converted to asparaginyl and glutaminyl residues byreaction with ammonium ions. Mimetics of basic amino acids can begenerated by substitution with, e.g., (in addition to lysine andarginine) the amino acids ornithine, citrulline, or (guanidino)-aceticacid, or (guanidino)alkyl-acetic acid, where alkyl is defined above.Nitrile derivative (e.g., containing the CN-moiety in place of COOH) canbe substituted for asparagine or glutamine. Asparaginyl and glutaminylresidues can be deaminated to the corresponding aspartyl or glutamylresidues. Arginine residue mimetics can be generated by reacting arginylwith, e.g., one or more conventional reagents, including, e.g.,phenylglyoxal, 2,3-butanedione, 1,2-cyclo-hexanedione, or ninhydrin,preferably under alkaline conditions. Tyrosine residue mimetics can begenerated by reacting tyrosyl with, e.g., aromatic diazonium compoundsor tetranitromethane. N-acetylimidizol and tetranitromethane can be usedto form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.Cysteine residue mimetics can be generated by reacting cysteinylresidues with, e.g., alpha-haloacetates such as 2-chloroacetic acid orchloroacetamide and corresponding amines; to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteine residue mimetics can also begenerated by reacting cysteinyl residues with, e.g.,bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic acid;chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide;methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4nitrophenol; or, chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimeticscan be generated (and amino terminal residues can be altered) byreacting lysinyl with, e.g., succinic or other carboxylic acidanhydrides. Lysine and other alpha-amino-containing residue mimetics canalso be generated by reaction with imidoesters, such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,trinitro-benzenesulfonic acid, O-methylisourea, 2,4, pentanedione, andtransamidase-catalyzed reactions with glyoxylate. Mimetics of methioninecan be generated by reaction with, e.g., methionine sulfoxide. Mimeticsof proline include, e.g., pipecolic acid, thiazolidine carboxylic acid,3- or 4-hydroxy proline, dehydroproline, 3- or 4-methylproline, or3,3,-dimethylproline. Histidine residue mimetics can be generated byreacting histidyl with, e.g., diethylprocarbonate or para-bromophenacylbromide. Other mimetics include, e.g., those generated by hydroxylationof proline and lysine; phosphorylation of the hydroxyl groups of serylor threonyl residues; methylation of the alpha-amino groups of lysine,arginine and histidine; acetylation of the N-terminal amine; methylationof main chain amide residues or substitution with N-methyl amino acids;or amidation of C-terminal carboxyl groups.

A residue, e.g., an amino acid, of a polypeptide of the invention canalso be replaced by an amino acid (or peptidomimetic residue) of theopposite chirality. Thus, any amino acid naturally occurring in theL-configuration (which can also be referred to as the R or S, dependingupon the structure of the chemical entity) can be replaced with theamino acid of the same chemical structural type or a peptidomimetic, butof the opposite chirality, referred to as the D-amino acid, but also canbe referred to as the R- or S-form.

The invention also provides methods for modifying the polypeptides ofthe invention by either natural processes, such as post-translationalprocessing (e.g., phosphorylation, acylation, etc), or by chemicalmodification techniques, and the resulting modified polypeptides.Modifications can occur anywhere in the polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini. It will be appreciated that the same type of modification maybe present in the same or varying degrees at several sites in a givenpolypeptide. Also a given polypeptide may have many types ofmodifications. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of a phosphatidylinositol, cross-linkingcyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristolyation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, and transfer-RNA mediatedaddition of amino acids to protein such as arginylation. See, e.g.,Creighton, T. E., Proteins—Structure and Molecular Properties 2nd Ed.,W.H. Freeman and Company, New York (1993); Posttranslational CovalentModification of Proteins, B. C. Johnson, Ed., Academic Press, New York,pp. 1-12 (1983).

Solid-phase chemical peptide synthesis methods can also be used tosynthesize the polypeptide or fragments of the invention. Such methodhave been known in the art since the early 1960's (Merrifield, R. B., J.Am. Chem. Soc., 85:2149-2154, 1963) (See also Stewart, J. M. and Young,J. D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co.,Rockford, Ill., pp. 11-12)) and have recently been employed incommercially available laboratory peptide design and synthesis kits(Cambridge Research Biochemicals). Such commercially availablelaboratory kits have generally utilized the teachings of H. M. Geysen etal, Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and provide forsynthesizing peptides upon the tips of a multitude of “rods” or “pins”all of which are connected to a single plate. When such a system isutilized, a plate of rods or pins is inverted and inserted into a secondplate of corresponding wells or reservoirs, which contain solutions forattaching or anchoring an appropriate amino acid to the pin's or rod'stips. By repeating such a process step, i.e., inverting and insertingthe rod's and pin's tips into appropriate solutions, amino acids arebuilt into desired peptides. In addition, a number of available FMOCpeptide synthesis systems are available. For example, assembly of apolypeptide or fragment can be carried out on a solid support using anApplied Biosystems, Inc. Model 431A™ automated peptide synthesizer. Suchequipment provides ready access to the peptides of the invention, eitherby direct synthesis or by synthesis of a series of fragments that can becoupled using other known techniques.

The invention provides novel amylases, including the exemplary enzymeshaving sequences as set forth in SEQ ID NO:2; SEQ ID NO:6; SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:16, nucleic acidsencoding them, antibodies that bind them, and methods for making andusing them. In one aspect, the polypeptides of the invention have anamylase activity, as described herein, including, e.g., the ability tohydrolyze starches into sugars. In alternative aspects, the amylases ofthe invention have activities that have been modified from those of theexemplary amylases described herein. The invention includes amylaseswith and without signal sequences and the signal sequences themselves.The invention includes immobilized amylases, anti-amylase antibodies andfragments thereof. The invention provides methods for inhibiting amylaseactivity, e.g., using dominant negative mutants or anti-amylaseantibodies of the invention. The invention includes heterocomplexes,e.g., fusion proteins, heterodimers, etc., comprising the amylases ofthe invention.

In one aspect, amylases of the invention hydrolyze internal α-1,4- and1,6-glucosidic bonds in starch to produce smaller molecular weightmaltodextrines. In one aspect, this hydrolysis is largely at random.Thus, the invention provides methods for producing smaller molecularweight maltodextrines.

Amylases of the invention can be used in laboratory and industrialsettings to hydrolyze starch or any maltodextrine-comprising compoundfor a variety of purposes. These amylases can be used alone to providespecific hydrolysis or can be combined with other amylases to provide a“cocktail” with a broad spectrum of activity. Exemplary uses include theremoval or partial or complete hydrolysis of starch or anymaltodextrine-comprising compound from biological, food, animal feed,pharmaceutical or industrial samples.

For example, the amylases of the present invention can be formulated inlaundry detergents to aid in the removal of starch-containing stains.Amylases of the invention can be used as cleaning agents in detergentmatrices (see industrial applications below). The amylases of thepresent invention can be used in the initial stages (liquefaction) ofstarch processing, in wet corn milling, in alcohol production, in thetextile industry for starch desizing, in baking applications, in thebeverage industry, in oilfields in drilling processes; in inking ofrecycled paper; and in animal feed.

Amylases of the invention can have an amylase activity under variousconditions, e.g., extremes in pH and/or temperature, oxidizing agents,and the like. The invention provides methods leading to alternativeamylase preparations with different catalytic efficiencies andstabilities, e.g., towards temperature, oxidizing agents and changingwash conditions. In one aspect, amylase variants can be produced usingtechniques of site-directed mutagenesis and/or random mutagenesis. Inone aspect, directed evolution can be used to produce a great variety ofamylase variants with alternative specificities and stability.

The proteins of the invention are also useful as research reagents toidentify amylase modulators, e.g., activators or inhibitors of amylaseactivity. Briefly, test samples (compounds, broths, extracts, and thelike) are added to amylase assays to determine their ability to inhibitsubstrate cleavage. Inhibitors identified in this way can be used inindustry and research to reduce or prevent undesired proteolysis. Aswith amylases, inhibitors can be combined to increase the spectrum ofactivity.

The invention also provides methods of discovering new amylases usingthe nucleic acids, polypeptides and antibodies of the invention. In oneaspect, lambda phage libraries are screened for expression-baseddiscovery of amylases. In one aspect, the invention uses lambda phagelibraries in screening to allow detection of toxic clones; improvedaccess to substrate; reduced need for engineering a host, by-passing thepotential for any bias resulting from mass excision of the library; and,faster growth at low clone densities. Screening of lambda phagelibraries can be in liquid phase or in solid phase. In one aspect, theinvention provides screening in liquid phase. This gives a greaterflexibility in assay conditions; additional substrate flexibility;higher sensitivity for weak clones; and ease of automation over solidphase screening.

The invention provides screening methods using the proteins and nucleicacids of the invention and robotic automation to enable the execution ofmany thousands of biocatalytic reactions and screening assays in a shortperiod of time, e.g., per day, as well as ensuring a high level ofaccuracy and reproducibility (see discussion of arrays, below). As aresult, a library of derivative compounds can be produced in a matter ofweeks. For further teachings on modification of molecules, includingsmall molecules, see PCT/US94/09174.

The present invention includes amylase enzymes which are non-naturallyoccurring carbonyl hydrolase variants (e.g., amylase variants) having adifferent proteolytic activity, stability, substrate specificity, pHprofile and/or performance characteristic as compared to the precursorcarbonyl hydrolase from which the amino acid sequence of the variant isderived. Specifically, such amylase variants have an amino acid sequencenot found in nature, which is derived by substitution of a plurality ofamino acid residues of a precursor amylase with different amino acids.The precursor amylase may be a naturally-occurring amylase or arecombinant amylase. The useful amylase variants encompass thesubstitution of any of the naturally occurring L-amino acids at thedesignated amino acid residue positions.

Exemplary SEQ ID NO:2 has the sequence:

Met Arg Val Ser Ser Ile Gly Asn Gly Arg Met LeuIle Asn Phe Asp Glu Lys Gly Arg Ile Val Asp IleTyr Tyr Pro Tyr Ile Gly Met Glu Asn Gln Thr SerGly Asn Pro Ile Arg Leu Ala Ile Trp Asp Lys AspLys Lys Val Ala Ser Leu Asp Glu Asp Trp Glu ThrThr Val Leu Tyr Ile Asp Glu Ala Asn Met Val GluIle Arg Ser Asp Val Lys Glu Leu Gly Leu Ser LeuLeu Ser Tyr Asn Phe Leu Asp Ser Asp Asp Pro IleTyr Met Ser Ile Val Lys Ile Ala Asn Asn Glu AsnAsn Ser Arg Asn Ile Lys Val Phe Phe Ile His AspIle Asn Leu Tyr Ser Asn Pro Phe Gly Asp Thr AlaPhe Tyr Asp Pro Leu Pro Leu Ser Ile Ile His TyrLys Ser Lys Arg Tyr Leu Ala Phe Lys Val Phe ThrThr Val Ser Thr Leu Ser Glu Tyr Asn Ile Gly LysGly Asp Leu Ile Gly Asp Ile Tyr Asp Gly Asn LeuGly Leu Asn Gly Ile Glu Asn Gly Asp Val Asn SerSer Met Gly Ile Glu Ile Asn Ile Asp Pro Asn SerTyr Leu Lys Leu Tyr Tyr Val Ile Val Ala Asp ArgAsn Leu Glu Gly Leu Arg Gln Lys Ile Arg Lys IleAsn Phe Ala Asn Val Glu Thr Ser Phe Thr Leu ThrTyr Met Phe Trp Arg Asn Trp Leu Lys Lys Asn LysLeu Phe Arg Asn Asn Leu Met Gln Asp Ile Lys ArgVal Tyr Asp Val Ser Leu Phe Val Ile Arg Asn HisMet Asp Val Asn Gly Ser Ile Ile Ala Ser Ser AspPhe Ser Phe Val Lys Ile Tyr Gly Asp Ser Tyr GlnTyr Cys Trp Pro Arg Asp Ala Ala Ile Ala Ala TyrAla Leu Asp Leu Ala Gly Tyr Lys Glu Leu Ala LeuLys His Phe Gln Phe Ile Ser Asn Ile Ala Asn SerGlu Gly Phe Leu Tyr His Lys Tyr Asn Pro Asn ThrThr Leu Ala Ser Ser Trp His Pro Trp Tyr Tyr LysGly Lys Arg Ile Tyr Pro Ile Gln Gly Asp Glu ThrAla Leu Glu Val Trp Ala Ile Ala Ser His Tyr GluLys Tyr Glu Asp Ile Asp Glu Ile Leu Pro Leu TyrLys Lys Phe Val Lys Pro Ala Leu Lys Phe Met MetSer Phe Met Glu Glu Gly Leu Pro Lys Pro Ser PheAsp Leu Trp Glu Glu Arg Tyr Gly Ile His Ile TyrThr Val Ser Thr Val Tyr Gly Ala Leu Thr Lys GlyAla Lys Leu Ala Tyr Asp Val Gly Asp Glu Ile LeuSer Glu Asp Leu Ser Asp Thr Ser Gly Leu Leu LysGly Met Val Leu Lys Arg Met Thr Tyr Asn Gly ArgPhe Val Arg Arg Ile Asp Glu Glu Asn Asn Gln AspLeu Thr Val Asp Ser Ser Leu Tyr Ala Pro Phe PhePhe Gly Leu Val Asn Ala Asn Asp Lys Ile Met IleAsn Thr Ile Asn Glu Ile Glu Ser Arg Leu Thr ValAsn Gly Gly Ile Ile Arg Tyr Glu Asn Asp Met TyrGln Arg Arg Lys Lys Gln Pro Asn Pro Trp Ile IleThr Thr Leu Trp Leu Ser Glu Tyr Tyr Ala Thr IleAsn Asp Lys Asn Lys Ala Asn Glu Tyr Ile Lys TrpVal Ile Asn Arg Ala Leu Pro Thr Gly Phe Leu ProGlu Gln Val Asp Pro Glu Thr Phe Glu Pro Thr SerVal Thr Pro Leu Val Trp Ser His Ala Glu Phe Ile Ile Ala Ile Asn Asn IleExemplary SEQ ID NO:4 has the sequence

Met Val Arg Tyr Thr Pro Leu Gly Asn Gly Arg LeuLeu Ile Ala Phe Asp Thr Asp Tyr Arg Ile Val AspPhe Tyr Tyr Ser Lys Phe Ala Ser Glu Asn His SerSer Gly His Pro Phe Tyr Phe Gly Val Ser Val AspGly Asn Phe Asn Trp Ile Asp Arg Asn Ala Ile LysHis Met Asp Tyr Tyr Asp His Thr Met Val Ser ValVal Asn Tyr Thr His Asn Gly Ile Asp Phe Glu AsnArg Asp Met Val Asp Ile Tyr Lys Asp Ile Phe IleArg Arg Val Val Ala Glu Asn Lys Thr Gly Lys AspVal Asn Leu Lys Ile Phe Phe His Gln Asn Phe TyrIle Tyr Gly Asn Asp Ile Gly Asp Thr Ala Ala TyrPhe Pro Glu Tyr Arg Gly Val Ile His Tyr Lys GlyGly Arg Tyr Phe Leu Ala Ser Thr Leu Asp Glu SerGly Asn Phe Cys Asp Gln Tyr Ala Thr Gly Val LysAsp Val Gly Glu Leu Lys Gly Thr Trp Lys Asp AlaGlu Asp Asn Glu Leu Ser Met Asn Pro Val Ala IleGly Ser Val Asp Ser Val Ile Arg His Ser Thr ThrLeu Lys Ala Gly Ser Lys Phe Thr Leu Tyr Tyr PheIle Ile Ala Gly Arg Asn Ile Asn Asp Ile Glu SerGlu Tyr Ser Asn Val Asn Val Gln Tyr Leu Gln LysLeu Leu Arg Arg Thr Thr Asn Tyr Trp Glu Leu TrpSer Ser Lys Val Thr Pro Ser Leu Asp Ser Asp ThrThr Ala Leu Tyr Arg Arg Ser Leu Phe Val Thr LysSer His Ala Asn Asp Leu Gly Ala Ile Ala Ala SerCys Asp Ser Asp Ile Leu Lys Leu Ser His Asp GlyTyr Tyr Tyr Val Trp Pro Arg Asp Ala Ser Met AlaAla Tyr Ala Leu Ser Ile Ser Gly His Ser Glu ThrAla Arg Arg Phe Phe Ala Leu Met Glu Asp Ser LeuSer Glu Glu Gly Tyr Leu Tyr His Lys Tyr Asn ValAsp Gly Lys Ile Ala Ser Ser Trp Leu Pro His ValMet Asn Gly Lys Ser Ile Tyr Pro Ile Gln Glu AspGlu Thr Ala Leu Val Val Trp Ala Leu Trp Glu TyrPhe Arg Lys Tyr Asn Asp Ile Gly Phe Thr Ala ProTyr Tyr Glu Arg Leu Ile Thr Arg Ala Ala Asp PheMet Thr Asn Phe Val Asp Asn Asn Gly Leu Pro LysPro Ser Phe Asp Leu Trp Glu Glu Arg Tyr Gly IleHis Ala Tyr Thr Val Ala Thr Val Tyr Ala Ala LeuLys Ala Ala Ser Asn Phe Ala Asn Val Phe Gly AspPro Asp Leu Ser Glu Lys Tyr Glu Asn Ala Ala GluArg Met Tyr His Ala Phe Asp Glu Arg Phe Tyr SerGlu Asp Thr Gly Tyr Tyr Ala Arg Ala Ile Ile AspGly Lys Pro Asp Phe Thr Val Asp Ser Ala Leu ThrSer Leu Val Leu Phe Gly Met Lys Asp Ala Asp AspPro Lys Val Ile Ser Thr Met Gln Arg Ile Ser GluAsp Leu Trp Val Asn Gly Val Gly Gly Ile Ala ArgTyr Gln Asn Asp Arg Tyr Met Arg Val Lys Asp AspPro Ser Val Pro Gly Asn Pro Trp Ile Ile Thr ThrLeu Trp Met Ala Arg Tyr Tyr Met Arg Phe Gly AspPhe Glu Lys Ala Trp Asn Leu Ile Gln Trp Val LysSer His Arg Gln Lys Ser Gly Ile Phe Ser Glu GlnIle Asn Pro Tyr Asn Gly Glu Pro Leu Ser Val SerPro Leu Val Trp Ser His Ser Glu Phe Ile Ile SerLeu Leu Glu Tyr Ser Asp Leu Ile Arg Asn Arg SerExemplary SEQ ID NO:6 has the sequence:

Met Ile Tyr Met Gly Gly Ile Ile Gly Asn Asn AsnLeu Leu Val Lys Ile Gly Asp Tyr Gly Glu Ile SerTyr Val Phe Tyr Pro His Val Gly Tyr Glu Thr HisPhe Phe Asp Ser Ala Leu Ala Val Tyr Asp Lys LysVal Lys Trp His Trp Asp Asp Asp Trp Asp Ile SerGln Lys Tyr Ile Glu Glu Thr Asn Ile Phe Lys ThrIle Leu Glu Asp Asp Lys Ile Ile Leu Thr Ile LysAsp Phe Val Pro Val Ser His Asn Val Ile Ile ArgArg Leu His Ile Lys Asn Lys Leu Asp Lys Lys LeuAsn Phe Lys Leu Phe Phe Tyr Glu Asn Leu Arg IleGly Glu Tyr Pro Thr Glu Asn Ala Val Arg Phe LeuGlu Asp Glu Gly Cys Ile Val Lys Tyr Asn Glu LysTyr Val Phe Cys Ile Gly Ser Asn Lys Lys Ile AspSer Phe Gln Cys Gly Asn Arg Tyr Ser Lys Asn SerAla Tyr Val Asp Ile Glu Asn Gly Leu Leu Met GluHis Lys Glu Ser His Gly Leu Met Thr Asp Ser AlaIle Ser Trp Asn Ile Glu Ile Asp Lys Gly Lys SerLeu Ala Phe Asn Ile Tyr Ile Leu Leu Gln Lys PheAsp Gly Asp Leu Ser Ile Ile Thr Glu Gln Leu LysIle Ile Met Asn Asn Thr Val His Ile Lys Asp LeuSer Met Asn Tyr Trp Lys Asn Ser Ile Gly Asn IleLys Glu His Ile His Pro Gln Phe His Ser Asp LysGlu Ile Cys Pro Ile Ala Lys Arg Ala Leu Met ValLeu Leu Met Leu Cys Asp Lys Asp Gly Gly Ile IleAla Ala Pro Ser Leu His Pro Asp Tyr Arg Tyr ValTrp Gly Arg Asp Gly Ala Tyr Ile Ala Ile Ala LeuAsp Leu Phe Gly Ile Arg Gly Ile Pro Asp Arg PhePhe Glu Phe Met Ser Lys Ile Gln Asn Asp Asp GlySer Trp Leu Gln Asn Tyr Tyr Thr Asn Gly Lys ProArg Leu Thr Ala Met Gln Ile Asp Gln Ile Gly SerIle Leu Trp Ala Met Asp Val His Tyr Arg Leu ThrGly Asn Arg Lys Phe Val Glu Arg Tyr Trp Asn ThrIle Glu Lys Ala Gly Asn Tyr Leu Thr Ser Ala AlaLeu Asn Phe Thr Pro Cys Phe Asp Leu Trp Glu GluLys Phe Gly Val Phe Ala Tyr Thr Met Gly Ala IleTyr Ala Gly Leu Lys Ala Ala Tyr Ser Met Ser LysAla Val Asp Met Arg Asp Lys Val Lys His Trp GluLys Ala Ile Glu Phe Leu Lys Lys Glu Val Pro ArgArg Phe Tyr Leu Glu Asp Glu Glu Arg Phe Ala LysSer Ile Asn Pro Leu Asp Lys Glu Ile Asp Ala SerIle Leu Gly Leu Ser Tyr Pro Phe Asn Leu Ile AspVal Asp Asp Glu Arg Met Ile Lys Thr Ala Glu AlaIle Glu Asn Ala Phe Asn Tyr Lys Val Gly Gly IleGly Arg Tyr Pro Asn Asp Val Tyr Phe Gly Gly AsnPro Trp Ile Ile Thr Thr Leu Trp Ile Ser Leu TyrTyr Arg Arg Leu Ser Lys Val Leu Lys Glu Lys AsnLys Asn Asp Met Ala Glu Lys Tyr Leu Lys Lys SerLys Lys Leu Phe Asp Trp Ala Val Lys Tyr Ser PheAsn Gly Leu Phe Pro Glu Gln Ile His Lys Asp LeuGly Ile Pro Met Ser Ala Met Pro Leu Gly Trp SerAsn Ala Met Phe Leu Ile Tyr Leu Tyr Lys Asp Asp Asn Val Ile Ile ProExemplary SEQ ID NO:8 has the sequence:

Met Ala Gly Ile Ile Gly Asn Gly Asn Leu Leu AlaLys Ile Asp Asp Leu Gly Ser Ile Glu Tyr Ile PhePhe Pro His Leu Gly Tyr Glu Thr His Ile Leu AspThr Ser Phe Ala Ile Tyr Tyr Asn Asn Lys Ile LysTrp His Trp Asp His Ser Trp Asp Val Ser Gln AsnTyr Leu Lys Asp Ser Asn Ile Leu Lys Thr Thr TyrGlu Asn Asp Asp Phe Leu Ile Tyr Ser Lys Asp CysVal Ser Ile Ser His Asn Leu Ile Val Lys Gln LeuSer Ile Ile Asn Lys Thr Asn Ser Glu Lys Asp IleLys Leu Phe Phe Tyr Glu Asn Leu Arg Ile Gly GluThr Pro Ser Lys Ser Thr Val Lys Phe Val Lys GluLys Asn Cys Leu Ile Lys His Asp Lys Asn Tyr IlePhe Cys Ile Gly Ser Asn Lys Lys Val Ser Ser TyrGln Cys Gly Ile Lys Tyr Ser Glu Ser Ser Ala LeuArg Asp Ile Glu Asn Gly Val Leu Lys Glu Gln SerSer Ala Thr Gly Leu Ile Thr Asp Ser Ala Leu CysTrp Glu Phe Lys Ile Lys Pro Asn Gln Lys Tyr ThrLeu Ser Ile Leu Ile Leu Pro Glu Lys Tyr Asp GlyAsp Tyr Asn Lys Thr Leu Asn Leu Met Asp Thr LeuHis Met Val Lys Asp Asn Leu Lys Asp Leu Tyr AsnLeu Thr Arg Asn Phe Trp Lys Ser Arg Val Asp SerMet Val Asn Lys Trp Gly Ile Leu Lys Leu Glu GluTyr Lys Glu Cys Ile Asp Ile Cys Lys Arg Ser LeuLeu Thr Leu Leu Leu Leu Cys Asp Tyr Lys Gly GlyIle Ile Ala Ser Pro Ser Leu His Pro Asp Tyr ArgTyr Val Trp Cys Arg Asp Ala Gly Tyr Met Ala ValAla Leu Asp Leu Cys Gly Gln His Glu Met Ser GluLys Tyr Phe Glu Trp Cys Lys Thr Thr Gln Asn SerAsp Gly Ser Trp Val Gln Asn Tyr Tyr Val Glu GlyTyr Pro Arg Phe Thr Ala Ile Gln Ile Asp Gln ValGly Thr Thr Ile Trp Ala Leu Leu Val His Tyr ArgIle Thr Gly Asp Lys His Phe Leu Lys Arg Asn TrpGlu Met Val Lys Lys Ala Gly Asp Tyr Leu Ser ArgAla Ala Asp Gln Leu Ile Pro Cys Tyr Asp Leu TrpGlu Glu Lys Phe Gly Val Phe Ala Tyr Thr Leu GlyAla Ile Tyr Gly Gly Leu Lys Ser Gly Tyr Leu IleGly Lys Glu Leu Asp Lys Glu Glu Glu Ile Gln HisTrp Lys Lys Ser Met Asn Phe Leu Lys Asn Glu ValVal Asn Arg Leu Tyr Leu Lys Asn Glu Lys Arg PheAla Lys Ser Leu Lys Pro Leu Asp Lys Thr Ile AspThr Ser Ile Leu Gly Leu Ser Phe Pro Tyr Gly LeuVal Ser Val Asp Asp Pro Arg Ile Ile Ser Thr AlaAsn Gln Ile Glu Lys Ala Phe Asn Tyr Lys Val GlyGly Val Gly Arg Tyr Pro Glu Asp Ile Tyr Phe GlyGly Asn Pro Trp Ile Ile Thr Thr Leu Trp Leu TyrMet Tyr Tyr Lys Lys Leu Val Asp Thr Leu Ser LysLys Gly Lys Phe Gln Glu Ser Ile Ile Asp Asn TyrAsn Lys Lys Cys Tyr Asn Leu Leu Lys Trp Ile LeuLys His Gln Phe Asn Gly Met Phe Pro Glu Gln ValHis Lys Asp Leu Gly Ile Pro Ile Ser Ala Ile ProLeu Gly Trp Ser His Ala Met Val Ile Ile Ala IleHis Gly Asp Tyr Asp Ile Leu Ile ProExemplary SEQ ID NO:10 has the sequence

Met Ile Tyr Met Gly Gly Ile Val Gly Asn Asn SerLeu Leu Ala Lys Ile Gly Asp Tyr Gly Glu Ile GluTyr Leu Phe Tyr Pro Gln Val Gly Tyr Glu Thr HisPhe Phe Asp Ser Ala Leu Ala Val Tyr Asp Lys LysVal Lys Trp His Trp Asp Asp Asp Trp Asp Ile ThrGln Lys Tyr Ile Glu Glu Thr Asn Ile Phe Lys ThrIle Leu Glu Asp Asp Lys Ile Ile Leu Thr Ile LysAsp Phe Val Pro Val Ser His Asn Val Leu Ile ArgArg Val Tyr Ile Lys Asn Lys Leu Asp Lys Lys LeuAsn Phe Lys Leu Phe Phe Tyr Glu Asn Leu Arg IleGly Glu Asn Pro Ile Thr Asn Thr Val Lys Phe LeuGlu Asp Gly Cys Ile Val Lys Tyr Asn Gly Lys TyrIle Phe Cys Ile Gly Ser Asp Lys Arg Ile Asp SerPhe Gln Cys Gly Asn Arg Tyr Ser Lys Thr Ser AlaTyr Ile Asp Ile Glu Asn Gly Ile Leu Lys Glu HisLys Glu Ser Ser Gly Leu Leu Thr Asp Ser Ala IleSer Trp Asn Ile Lys Ile Asp Glu Lys Arg Ser LeuAla Phe Asn Ile Tyr Ile Leu Pro Gln Arg Phe AspGly Asp Phe Ser Ile Ile Thr Glu Gln Leu Lys IleIle Met Asn Asn Ser Glu Asn Ile Lys Asn Leu SerMet Asn Tyr Trp Lys His Ile Ile Gly Glu Ile AsnArg Phe Ile His Pro Glu Leu Arg Gln Asn Asn LysIle Tyr Ser Ile Thr Lys Arg Ala Leu Met Thr LeuLeu Met Leu Cys Asp Lys Glu Gly Gly Ile Ile AlaAla Pro Ser Leu His Pro Asp Tyr Arg Tyr Val TrpGly Arg Asp Gly Ser Tyr Ile Ser Ile Ala Leu AspLeu Phe Gly Ile Arg Asn Ile Pro Asp Arg Phe PheGlu Phe Met Ser Lys Ile Gln Asn Ala Asp Gly SerTrp Leu Gln Asn Tyr Tyr Val Asn Gly Lys Pro ArgLeu Thr Ala Ile Gln Thr Asp Gln Ile Gly Ser IleLeu Trp Ala Met Asp Val His Tyr Arg Leu Thr GlyAsp Arg Lys Phe Val Glu Arg Tyr Trp Asn Thr IleGlu Lys Ala Ala Asn Tyr Leu Arg Leu Val Ala LeuAsn Phe Thr Pro Cys Phe Asp Leu Trp Glu Glu ArgPhe Gly Val Phe Ala Tyr Thr Met Gly Ala Thr TyrAla Gly Leu Lys Cys Ala Tyr Ser Met Ser Lys AlaVal Asn Lys Arg Asp Lys Val Lys Asp Trp Gly LysThr Ile Glu Phe Leu Lys His Glu Val Pro Lys ArgPhe Tyr Leu Glu Asp Glu Glu Arg Phe Ala Lys SerIle Asn Pro Leu Asp Lys Thr Ile Asp Thr Ser IleLeu Gly Leu Ser Tyr Pro Phe Asn Leu Ile Asp ValAsp Asp Glu Arg Met Ile Lys Thr Ala Glu Ala IleGlu Lys Ala Phe Lys Tyr Lys Val Gly Gly Ile GlyArg Tyr Pro Glu Asp Ile Tyr Phe Gly Gly Asn ProTrp Ile Ile Thr Thr Leu Trp Leu Ser Leu Tyr TyrArg Arg Leu Tyr Lys Val Leu Lys Glu Lys Asp AspAsn Gly Ala Asp Ile Tyr Leu Gln Leu Phe Pro GluGln Ile His Lys Glu Leu Gly Val Pro Met Ser AlaMet Pro Leu Gly Trp Ser Asn Ala Met Phe Leu IleTyr Val Tyr Glu Asn Asp Lys Val Ile Ile ProExemplary SEQ ID NO:12 has the sequence

Met Val Ser Met Val Gly Ile Ile Gly Asn Gly LysIle Leu Ala Lys Ile Asp Asp Ser Gly Ser Leu GluTyr Ile Phe Phe Pro His Leu Gly His Glu Lys HisIle Phe Asp Ser Ser Phe Ala Ile Phe Tyr Asp AsnLys Leu Lys Trp Asn Trp Asp Asn Ser Trp Asp IleAsn Gln Asn Tyr Leu Lys Asp Thr Asn Ile Leu LysThr Ser Tyr Glu Asn Glu Asp Phe Leu Ile Glu SerLys Asp Tyr Val Pro Ile Ser His Asn Ser Ile IleLys Gln Ile Ser Ile Leu Asn Lys Ser Ser Glu LysLys Asn Leu Lys Leu Phe Phe Tyr Glu Asn Leu ArgMet Gly Glu Ile Pro Glu Val Ser Thr Val Lys TyrArg Lys Asn Arg Glu Cys Ile Ile Lys Tyr Asp LysAsn Tyr Val Phe Cys Ile Gly Ser Asn Lys Lys ValSer Ser Tyr Gln Cys Gly Val Arg Ser Ser Glu SerSer Ala Leu Asn Asp Leu Lys Asn Gly Ile Leu LysGlu Tyr Asp Ser Ala Glu Gly Leu Ile Thr Asp SerAla Leu Gly Trp Asp Leu Glu Leu Ser Pro Asn GlnGlu Gln Lys Val Ser Ile Phe Ile Phe Ala Asp LysTyr Gly Gly Asp Tyr Thr Lys Ile Met Asn Leu LeuAsp Thr Leu Asn Ile Val Ile Thr Asn His Ala AspIle Tyr Asp Leu Thr Met Ala Tyr Trp Lys Asn MetIle Glu Thr Thr Ala Asn Ser Leu Cys Asn Ser AsnGln Val Phe Lys Asp Leu Thr His Ile Lys Asp AspAla Asn Ile Ser Asn Leu Lys Arg Ile Lys Gln TyrGlu Ala Ile Cys Lys Arg Ser Leu Leu Thr Ile LeuLeu Leu Cys Asp His Asn Gly Gly Ile Ile Ala SerPro Ser Leu Tyr Pro Asp Tyr Arg Tyr Val Trp CysArg Asp Ala Gly Tyr Met Ala Val Ala Leu Asp LeuCys Gly Gln His Gly Ile Ser Glu Lys Tyr Phe GluTrp Cys Lys Lys Thr Gln Asn Ser Asp Gly Ser TrpVal Gln Asn Tyr Tyr Val Glu Gly Asn Pro Arg LeuThr Ala Ile Gln Ile Asp Gln Val Gly Thr Thr IleTrp Ala Ala Leu Val His Tyr Arg Ile Thr Arg AspLys Leu Phe Leu Asn Arg Tyr Trp Glu Met Ile LysLys Ala Gly Asp Tyr Leu Ser Ser Val Ala Asn ProPro Ser Pro Ser Tyr Asp Leu Trp Glu Glu Lys TyrGly Val Phe Ala Tyr Thr Leu Gly Ala Ile Tyr GlyGly Leu Lys Ser Ala Tyr Asn Ile Cys Lys Ile LeuGly Lys Glu Glu His Asp Ile Gln Asn Trp Lys GluSer Met Asp Phe Leu Lys Asn Glu Met Val Asp ArgLeu Tyr Leu Lys Asp Glu Asn Arg Phe Ala Lys SerLeu Asp Pro Leu Asp Lys Ala Leu Asp Ala Ser IleLeu Gly Leu Ser Phe Pro Tyr Asn Leu Val Pro ValAsp Asp Pro Arg Met Ile Ser Thr Ala Asn Gln IleGlu Asn Ala Phe Lys Tyr Lys Val Gly Gly Ile GlyArg Tyr Pro Glu Asp Val Tyr Phe Gly Gly Asn ProTrp Ile Ile Thr Thr Ile Trp Leu His Met Tyr TyrGlu Asn Leu Ile Lys Ser Leu Ser Lys His Gly LysAsn Ala Ile His Ser Asp Gln Ile Pro Asp Ser SerGly Asp Leu Lys Asp Phe Val Ser Ile Ile Gly SerIle Glu Asn His Gly Glu Lys Ser Asp Glu Thr ProSer Ser Asp Thr Leu Leu Thr Tyr Ala Gln Lys CysAsn Asn Leu Phe Asp Trp Thr Leu Lys Tyr Asn PheAsn Glu Leu Phe Pro Glu Gln Val His Lys Asp LeuGly Ala Pro Ile Ser Ala Ile Pro Leu Gly Trp SerHis Ala Met Val Ile Ile Ala Ile His Gly Asn Phe Asp Ile Leu Ile ProExemplary SEQ ID NO:14 has the sequence:

Met Ile Val Gly Asn Asn Ser Phe Leu Cys Lys IleGly Asp His Gly Glu Ile Glu Tyr Ala Phe Tyr Pro His Val Gly Tyr Glu Leu His Phe Phe Asp Ser SerLeu Ala Ile Tyr Asp Lys Glu Ile Met Trp Ile TrpAsp Lys Glu Trp Ser Val Tyr Gln Lys Tyr Ile GluAsp Thr Asn Ile Phe Lys Thr Thr Leu Glu Asn GluAsn Ile Ile Phe Val Ile Lys Asp Leu Val Pro IleSer His Asn Val Leu Ile Arg Arg Val Phe Ile LysAsn Lys Leu Pro Tyr Asn Tyr Asn Phe Lys Leu PhePhe Tyr Glu Asn Leu Arg Ile Gly Glu His Pro SerGlu Asn Thr Val Lys Phe Leu Asp Asp Cys Ile ValLys Phe Asn Gly Lys Tyr Thr Phe Cys Ile Ser SerAsp Lys Lys Ile Asn Ser Tyr Gln Cys Gly Asn ArgTyr Ser Glu Lys Ser Ala Tyr Lys Asp Ile Glu AsnGly Leu Leu Ser Glu Asn Pro Glu Ser Val Gly ValLeu Thr Asp Ser Ala Ile Glu Trp Asp Ile Asp LeuLys Pro His Gly Lys Val Ala Phe Asn Ile Tyr IlePhe Pro His Ile Gly Asn Asn Ile Glu Ile Ile LysAsn Gln Leu Asn Ile Ile Lys Asn Leu Ser Ser GluIle Lys Asn Ile Ser Leu Asn Tyr Trp Lys Ser SerPhe Asp Ile Lys Gly Tyr Leu Phe Asn Glu Lys TyrLeu Lys Leu Ala Lys Arg Ala Leu Met Ile Leu ThrMet Leu Ser Asp Lys Asn Gly Gly Ile Ile Ala SerPro Ser Ile His Pro Asp Tyr Arg Tyr Val Trp GlyArg Asp Gly Ser Tyr Met Ala Val Ala Leu Ser IleTyr Gly Ile Lys Asn Ile Pro Trp Arg Phe Phe HisPhe Met Ser Lys Val Gln Asn Leu Asp Gly Ser TrpLeu Gln Asn Tyr Tyr Thr Asp Gly Lys Pro Arg LeuThr Ala Leu Gln Ile Asp Gln Ile Gly Ser Val LeuTrp Ala Met Glu Val Tyr Tyr Arg Thr Thr Gly AspArg Glu Phe Val Lys Lys Phe Trp Glu Thr Ile GluLys Ala Gly Asn Phe Leu Tyr Asn Ala Ser Leu SerLeu Met Pro Cys Phe Asp Leu Trp Glu Glu Lys TyrGly Val Phe Ser Tyr Thr Leu Gly Ala Met Tyr GlyGly Leu Arg Ala Gly Cys Ser Leu Ala Lys Ala IleGlu Glu Lys Lys Glu Asp Trp Lys Lys Ala Leu AspLys Leu Lys Lys Asp Val Asp Leu Leu Tyr Leu SerAsp Glu Glu Arg Phe Val Lys Ser Ile Asn Pro LeuAsn Lys Glu Ile Asp Thr Ser Ile Leu Gly Leu SerTyr Pro Phe Gly Leu Val Lys Val Asn Asp Glu ArgMet Ile Lys Thr Ala Glu Ala Ile Glu Lys Ala PheLys Tyr Lys Val Gly Gly Ile Gly Arg Tyr Pro SerAsp Val Tyr Phe Gly Gly Asn Pro Trp Ile Ile ThrThr Leu Trp Leu Ala Leu Tyr Tyr Arg Arg Leu PheIle Thr Thr Asn Asp Arg Lys Tyr Leu Glu Lys SerLys Lys Leu Phe Asn Trp Val Ile Asn His Ile TyrLeu Phe Pro Glu Gln Ile His Lys Glu Leu Ala IlePro Val Ser Ala Met Pro Leu Gly Trp Ser Cys AlaMet Leu Leu Phe Tyr Leu Tyr Lys Asn Asp Asp Ile Ile Val Ile LysExemplary SEQ ID NO:16 has the sequence:

Met Lys Leu Asn Arg Lys Leu Ile Lys Tyr Leu ProVal Leu Phe Leu Ala Ser Ser Val Leu Ser Gly CysAla Asn Asn Asn Ile Ser Asn Ile Lys Ile Glu ArgLeu Asn Asn Val Gln Ala Val Asn Gly Pro Gly GluAla Asp Thr Trp Ala Lys Ala Gln Lys Gln Gly ValGly Thr Ala Asn Asn Tyr Thr Ser Lys Val Trp PheThr Ile Ala Asp Gly Gly Ile Ser Glu Val Tyr TyrPro Thr Ile Asp Thr Ala Asp Val Lys Asp Ile LysPhe Phe Val Thr Asp Gly Lys Thr Phe Val Ser AspGlu Thr Lys Asp Thr Ile Thr Lys Val Glu Lys PheThr Glu Lys Ser Leu Gly Tyr Lys Ile Ile Asn ThrAsp Lys Glu Gly Arg Tyr Lys Ile Thr Lys Glu IlePhe Thr Asp Val Lys Arg Asn Ser Leu Val Ile LysThr Lys Phe Glu Ala Leu Lys Gly Asn Val Asp AspTyr Arg Leu Tyr Val Met Cys Asp Pro His Val LysAsn Gln Gly Lys Tyr Asn Glu Gly Tyr Ala Val LysAla Asn Gly Asn Val Ala Leu Ile Ala Glu Arg AspGly Ile Tyr Thr Ala Leu Ser Ser Asp Ile Gly TrpLys Lys Tyr Ser Ile Gly Tyr Tyr Lys Val Asn AspIle Glu Thr Asp Leu Tyr Lys Asn Met Gln Met ThrTyr Asn Tyr Asp Ser Ala Arg Gly Asn Ile Ile GluGly Ala Glu Ile Asp Leu Lys Lys Asn Arg Gln PheGlu Ile Val Leu Ser Phe Gly Gln Ser Glu Asp GluAla Val Lys Thr Asn Met Glu Thr Leu Asn Asp AsnTyr Asp Ser Leu Lys Lys Ala Tyr Ile Asp Gln TrpGlu Lys Tyr Cys Asp Ser Leu Asn Asp Phe Gly GlyLys Ala Asn Ser Leu Tyr Phe Asn Ser Met Met IleLeu Lys Ala Ser Glu Asp Lys Thr Asn Lys Gly AlaTyr Ile Ala Ser Leu Ser Ile Pro Trp Gly Asp GlyGln Glu Asp Asp Asn Ile Gly Gly Tyr His Leu ValTrp Ser Arg Asp Leu Tyr His Val Ala Asn Ala PheIle Val Ala Gly Asp Thr Asp Ser Ala Asn Arg AlaLeu Asp Tyr Leu Asp Lys Val Val Lys Asp Asn GlyMet Ile Pro Gln Asn Thr Trp Ile Asn Gly Arg ProTyr Trp Thr Gly Ile Gln Leu Asp Glu Gln Ala AspPro Ile Ile Leu Ser Tyr Arg Leu Lys Arg Tyr AspLeu Tyr Glu Ser Leu Val Lys Pro Leu Ala Asp PheIle Met Lys Ile Gly Pro Lys Thr Gly Gln Glu ArgTrp Glu Glu Ile Gly Gly Tyr Ser Pro Ala Thr LeuAla Ser Glu Val Ala Gly Leu Thr Cys Ala Ala TyrIle Ala Glu Gln Asn Lys Asp Phe Glu Ser Ala LysLys Tyr Gln Glu Lys Ala Asp Asn Trp Gln Arg LeuIle Asp Asn Leu Thr Tyr Thr Glu Lys Gly Pro LeuGly Asp Gly His Tyr Tyr Ile Arg Ile Ala Gly LeuPro Asp Pro Asn Ala Asp Phe Met Ile Ser Ile AlaAsn Gly Gly Gly Val Tyr Asp Gln Lys Glu Ile ValAsp Pro Ser Phe Leu Glu Leu Val Arg Leu Gly ValLys Ser Ala Asp Asp Pro Lys Ile Leu Asn Thr LeuLys Val Val Asp Glu Thr Ile Lys Val Asp Thr ProLys Gly Pro Ser Trp Tyr Arg Tyr Asn His Asp GlyTyr Gly Glu Met Ser Lys Thr Glu Leu Tyr His GlyThr Gly Lys Gly Arg Leu Trp Pro Leu Leu Thr GlyGlu Arg Gly Met Tyr Glu Ile Ala Ala Glu Tyr AspAsp Val Ile Ile Ile Lys Thr Arg Ile Gly Leu LeuLys Gly Ser Arg Ile Arg Phe Glu Tyr Asp Ile ValLys Glu Asp Glu Asn Lys Leu Leu Ala Gln Gly MetThr Glu His Pro Phe Thr Thr Leu Asp Arg Lys ProVal Asn Ile Lys Lys Ile Leu Pro His Val Tyr GluMet Leu Asn Lys Cys Tyr Asp Asp Gly Val

Amylase Signal Sequences

The invention also provides amylase-encoding nucleic acids comprisingsignal sequences. In one aspect, the signal sequences of the inventionare identified following identification of novel amylase polypeptides.

The pathways by which proteins are sorted and transported to theirproper cellular location are often referred to as protein targetingpathways. One of the most important elements in all of these targetingsystems is a short amino acid sequence at the amino terminus of a newlysynthesized polypeptide called the signal sequence. This signal sequencedirects a protein to its appropriate location in the cell and is removedduring transport or when the protein reaches its final destination. Mostlysosomal, membrane, or secreted proteins have an amino-terminal signalsequence that marks them for translocation into the lumen of theendoplasmic reticulum. More than 100 signal sequences for proteins inthis group have been determined. The sequences vary in length from 13 to36 amino acid residues. Various methods of recognition of signalsequences are known to those of skill in the art. For example, in oneaspect, novel amylase signal peptides are identified by a methodreferred to as SignalP. SignalP uses a combined neural network whichrecognizes both signal peptides and their cleavage sites. (Nielsen, etal., “Identification of prokaryotic and eukaryotic signal peptides andprediction of their cleavage sites.” Protein Engineering, vol. 10, no.1, p. 1-6 (1997).

It should be understood that in some aspects amylases of the inventionmay not have signal sequences. It may be desirable to include a nucleicacid sequence encoding a signal sequence from one amylase operablylinked to a nucleic acid sequence of a different amylase or, optionally,a signal sequence from a non-amylase protein may be desired. Table 3shows signal sequences of the invention.

Amylase Signal Sequences, Prepro and Catalytic Domains

The invention provides amylase signal sequences (e.g., signal peptides(SPs)), prepro domains and catalytic domains (CDs). The SPs, preprodomains and/or CDs of the invention can be isolated or recombinantpeptides or can be part of a fusion protein, e.g., as a heterologousdomain in a chimeric protein. The invention provides nucleic acidsencoding these catalytic domains (CDs), prepro domains and signalsequences (SPs, e.g., a peptide having a sequence comprising/consistingof amino terminal residues of a polypeptide of the invention).

In one aspect, the invention provides a signal sequence comprising apeptide comprising/consisting of a sequence as set forth in residues 1to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1to 28, 1 to 28, 1 to 30, 1 to 31, 1 to 32, 1 to 33, 1 to 34, 1 to 35, 1to 36, 1 to 37, 1 to 38, 1 to 39, 1 to 40, 1 to 41, 1 to 42, 1 to 43, 1to 44 (or a longer peptide) of a polypeptide of the invention.

The amylase signal sequences (SPs) and/or prepro sequences of theinvention can be isolated peptides, or, sequences joined to anotherprotease or a non-protease polypeptide, e.g., as a fusion (chimeric)protein. In one aspect, the invention provides polypeptides comprisingamylase signal sequences of the invention. In one aspect, polypeptidescomprising amylase signal sequences SPs and/or prepro of the inventioncomprise sequences heterologous to an amylase of the invention (e.g., afusion protein comprising an SP and/or prepro of the invention andsequences from another amylase or a non-amylase protein). In one aspect,the invention provides amylases of the invention with heterologous SPsand/or prepro sequences, e.g., sequences with a yeast signal sequence.An amylase of the invention can comprise a heterologous SP and/or preproin a vector, e.g., a pPIC series vector (Invitrogen, Carlsbad, Calif.).

In one aspect, SPs and/or prepro sequences of the invention areidentified following identification of novel amylase polypeptides. Thepathways by which proteins are sorted and transported to their propercellular location are often referred to as protein targeting pathways.One of the most important elements in all of these targeting systems isa short amino acid sequence at the amino terminus of a newly synthesizedpolypeptide called the signal sequence. This signal sequence directs aprotein to its appropriate location in the cell and is removed duringtransport or when the protein reaches its final destination. Mostlysosomal, membrane, or secreted proteins have an amino-terminal signalsequence that marks them for translocation into the lumen of theendoplasmic reticulum. More than 100 signal sequences for proteins inthis group have been determined. The signal sequences can vary in lengthfrom 13 to 36 or more amino acid residues. Various methods ofrecognition of signal sequences are known to those of skill in the art.For example, in one aspect, novel amylase signal peptides are identifiedby a method referred to as SignalP. SignalP uses a combined neuralnetwork which recognizes both signal peptides and their cleavage sites.(Nielsen, et al., “Identification of prokaryotic and eukaryotic signalpeptides and prediction of their cleavage sites.” Protein Engineering,vol. 10, no. 1, p. 1-6 (1997).

It should be understood that in some aspects amylases of the inventionmay not have SPs and/or prepro sequences, or “domains.” In one aspect,the invention provides the amylases of the invention lacking all or partof an SP and/or a prepro domain. In one aspect, the invention provides anucleic acid sequence encoding a signal sequence (SP) and/or prepro fromone amylase operably linked to a nucleic acid sequence of a differentamylase or, optionally, a signal sequence (SPs) and/or prepro domainfrom a non-amylases protein may be desired.

The invention also provides isolated or recombinant polypeptidescomprising signal sequences (SPs), prepro domain and/or catalyticdomains (CDs) of the invention and heterologous sequences. Theheterologous sequences are sequences not naturally associated (e.g., toan amylase) with an SP, prepro domain and/or CD. The sequence to whichthe SP, prepro domain and/or CD are not naturally associated can be onthe SP's, prepro domain and/or CD's amino terminal end, carboxy terminalend, and/or on both ends of the SP and/or CD. In one aspect, theinvention provides an isolated or recombinant polypeptide comprising (orconsisting of) a polypeptide comprising a signal sequence (SP), preprodomain and/or catalytic domain (CD) of the invention with the provisothat it is not associated with any sequence to which it is naturallyassociated (e.g., an amylase sequence). Similarly in one aspect, theinvention provides isolated or recombinant nucleic acids encoding thesepolypeptides. Thus, in one aspect, the isolated or recombinant nucleicacid of the invention comprises coding sequence for a signal sequence(SP), prepro domain and/or catalytic domain (CD) of the invention and aheterologous sequence (i.e., a sequence not naturally associated withthe a signal sequence (SP), prepro domain and/or catalytic domain (CD)of the invention). The heterologous sequence can be on the 3′ terminalend, 5′ terminal end, and/or on both ends of the SP, prepro domainand/or CD coding sequence.

Hybrid Amylases and Peptide Libraries

In one aspect, the invention provides hybrid amylases and fusionproteins, including peptide libraries, comprising sequences of theinvention. The peptide libraries of the invention can be used to isolatepeptide modulators (e.g., activators or inhibitors) of targets, such asamylase substrates, receptors, enzymes. The peptide libraries of theinvention can be used to identify formal binding partners of targets,such as ligands, e.g., cytokines, hormones and the like.

In one aspect, the fusion proteins of the invention (e.g., the peptidemoiety) are conformationally stabilized (relative to linear peptides) toallow a higher binding affinity for targets. The invention providesfusions of amylases of the invention and other peptides, including knownand random peptides. They can be fused in such a manner that thestructure of the amylases is not significantly perturbed and the peptideis metabolically or structurally conformationally stabilized. Thisallows the creation of a peptide library that is easily monitored bothfor its presence within cells and its quantity.

Amino acid sequence variants of the invention can be characterized by apredetermined nature of the variation, a feature that sets them apartfrom a naturally occurring form, e.g., an allelic or interspeciesvariation of an amylase sequence. In one aspect, the variants of theinvention exhibit the same qualitative biological activity as thenaturally occurring analogue. Alternatively, the variants can beselected for having modified characteristics. In one aspect, while thesite or region for introducing an amino acid sequence variation ispredetermined, the mutation per se need not be predetermined. Forexample, in order to optimize the performance of a mutation at a givensite, random mutagenesis may be conducted at the target codon or regionand the expressed amylase variants screened for the optimal combinationof desired activity. Techniques for making substitution mutations atpredetermined sites in DNA having a known sequence are well known, asdiscussed herein for example, M13 primer mutagenesis and PCRmutagenesis. Screening of the mutants can be done using assays ofproteolytic activities. In alternative aspects, amino acid substitutionscan be single residues; insertions can be on the order of from about 1to 20 amino acids, although considerably larger insertions can be done.Deletions can range from about 1 to about 20, 30, 40, 50, 60, 70residues or more. To obtain a final derivative with the optimalproperties, substitutions, deletions, insertions or any combinationthereof may be used. Generally, these changes are done on a few aminoacids to minimize the alteration of the molecule. However, largerchanges may be tolerated in certain circumstances.

The invention provides amylases where the structure of the polypeptidebackbone, the secondary or the tertiary structure, e.g., analpha-helical or beta-sheet structure, has been modified. In one aspect,the charge or hydrophobicity has been modified. In one aspect, the bulkof a side chain has been modified. Substantial changes in function orimmunological identity are made by selecting substitutions that are lessconservative. For example, substitutions can be made which moresignificantly affect: the structure of the polypeptide backbone in thearea of the alteration, for example a alpha-helical or a beta-sheetstructure; a charge or a hydrophobic site of the molecule, which can beat an active site; or a side chain. The invention provides substitutionsin polypeptide of the invention where (a) a hydrophilic residues, e.g.seryl or threonyl, is substituted for (or by) a hydrophobic residue,e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g. lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g. glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.phenylalanine, is substituted for (or by) one not having a side chain,e.g. glycine. The variants can exhibit the same qualitative biologicalactivity (i.e. amylase activity) although variants can be selected tomodify the characteristics of the amylases as needed.

In one aspect, amylases of the invention comprise epitopes orpurification tags, signal sequences or other fusion sequences, etc. Inone aspect, the amylases of the invention can be fused to a randompeptide to form a fusion polypeptide. By “fused” or “operably linked”herein is meant that the random peptide and the amylase are linkedtogether, in such a manner as to minimize the disruption to thestability of the amylase structure, e.g., it retains amylase activity.The fusion polypeptide (or fusion polynucleotide encoding the fusionpolypeptide) can comprise further components as well, including multiplepeptides at multiple loops.

In one aspect, the peptides and nucleic acids encoding them arerandomized, either fully randomized or they are biased in theirrandomization, e.g. in nucleotide/residue frequency generally or perposition. “Randomized” means that each nucleic acid and peptide consistsof essentially random nucleotides and amino acids, respectively. In oneaspect, the nucleic acids which give rise to the peptides can bechemically synthesized, and thus may incorporate any nucleotide at anyposition. Thus, when the nucleic acids are expressed to form peptides,any amino acid residue may be incorporated at any position. Thesynthetic process can be designed to generate randomized nucleic acids,to allow the formation of all or most of the possible combinations overthe length of the nucleic acid, thus forming a library of randomizednucleic acids. The library can provide a sufficiently structurallydiverse population of randomized expression products to affect aprobabilistically sufficient range of cellular responses to provide oneor more cells exhibiting a desired response. Thus, the inventionprovides an interaction library large enough so that at least one of itsmembers will have a structure that gives it affinity for some molecule,protein, or other factor.

Screening Methodologies and “On-Line” Monitoring Devices

In practicing the methods of the invention, a variety of apparatus andmethodologies can be used to in conjunction with the polypeptides andnucleic acids of the invention, e.g., to screen polypeptides for amylaseactivity, to screen compounds as potential modulators, e.g., activatorsor inhibitors, of an amylase activity, for antibodies that bind to apolypeptide of the invention, for nucleic acids that hybridize to anucleic acid of the invention, to screen for cells expressing apolypeptide of the invention and the like.

Capillary Arrays

Capillary arrays, such as the GIGAMATRIX™, Diversa Corporation, SanDiego, Calif., can be used to in the methods of the invention. Nucleicacids or polypeptides of the invention can be immobilized to or appliedto an array, including capillary arrays. Arrays can be used to screenfor or monitor libraries of compositions (e.g., small molecules,antibodies, nucleic acids, etc.) for their ability to bind to ormodulate the activity of a nucleic acid or a polypeptide of theinvention. Capillary arrays provide another system for holding andscreening samples. For example, a sample screening apparatus can includea plurality of capillaries formed into an array of adjacent capillaries,wherein each capillary comprises at least one wall defining a lumen forretaining a sample. The apparatus can further include interstitialmaterial disposed between adjacent capillaries in the array, and one ormore reference indicia formed within of the interstitial material. Acapillary for screening a sample, wherein the capillary is adapted forbeing bound in an array of capillaries, can include a first walldefining a lumen for retaining the sample, and a second wall formed of afiltering material, for filtering excitation energy provided to thelumen to excite the sample.

A polypeptide or nucleic acid, e.g., a ligand, can be introduced into afirst component into at least a portion of a capillary of a capillaryarray. Each capillary of the capillary array can comprise at least onewall defining a lumen for retaining the first component. An air bubblecan be introduced into the capillary behind the first component. Asecond component can be introduced into the capillary, wherein thesecond component is separated from the first component by the airbubble. A sample of interest can be introduced as a first liquid labeledwith a detectable particle into a capillary of a capillary array,wherein each capillary of the capillary array comprises at least onewall defining a lumen for retaining the first liquid and the detectableparticle, and wherein the at least one wall is coated with a bindingmaterial for binding the detectable particle to the at least one wall.The method can further include removing the first liquid from thecapillary tube, wherein the bound detectable particle is maintainedwithin the capillary, and introducing a second liquid into the capillarytube.The capillary array can include a plurality of individual capillariescomprising at least one outer wall defining a lumen. The outer wall ofthe capillary can be one or more walls fused together. Similarly, thewall can define a lumen that is cylindrical, square, hexagonal or anyother geometric shape so long as the walls form a lumen for retention ofa liquid or sample. The capillaries of the capillary array can be heldtogether in close proximity to form a planar structure. The capillariescan be bound together, by being fused (e.g., where the capillaries aremade of glass), glued, bonded, or clamped side-by-side. The capillaryarray can be formed of any number of individual capillaries, forexample, a range from 100 to 4,000,000 capillaries. A capillary arraycan form a micro titer plate having about 100,000 or more individualcapillaries bound together.

Arrays, or “Biochips”

Nucleic acids or polypeptides of the invention can be immobilized to orapplied to an array. Arrays can be used to screen for or monitorlibraries of compositions (e.g., small molecules, antibodies, nucleicacids, etc.) for their ability to bind to or modulate the activity of anucleic acid or a polypeptide of the invention. For example, in oneaspect of the invention, a monitored parameter is transcript expressionof an amylase gene. One or more, or, all the transcripts of a cell canbe measured by hybridization of a sample comprising transcripts of thecell, or, nucleic acids representative of or complementary totranscripts of a cell, by hybridization to immobilized nucleic acids onan array, or “biochip.” By using an “array” of nucleic acids on amicrochip, some or all of the transcripts of a cell can besimultaneously quantified. Alternatively, arrays comprising genomicnucleic acid can also be used to determine the genotype of a newlyengineered strain made by the methods of the invention. Polypeptidearrays” can also be used to simultaneously quantify a plurality ofproteins. The present invention can be practiced with any known “array,”also referred to as a “microarray” or “nucleic acid array” or“polypeptide array” or “antibody array” or “biochip,” or variationthereof. Arrays are generically a plurality of “spots” or “targetelements,” each target element comprising a defined amount of one ormore biological molecules, e.g., oligonucleotides, immobilized onto adefined area of a substrate surface for specific binding to a samplemolecule, e.g., mRNA transcripts.

In practicing the methods of the invention, any known array and/ormethod of making and using arrays can be incorporated in whole or inpart, or variations thereof, as described, for example, in U.S. Pat.Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695;6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174;5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522;5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; see also, e.g.,WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; see also, e.g.,Johnston (1998) Curr. Biol. 8:R171-R174; Schummer (1997) Biotechniques23:1087-1092; Kern (1997) Biotechniques 23:120-124; Solinas-Toldo (1997)Genes, Chromosomes & Cancer 20:399-407; Bowtell (1999) Nature GeneticsSupp. 21:25-32. See also published U.S. patent applications Nos.20010018642; 20010019827; 20010016322; 20010014449; 20010014448;20010012537; 20010008765.

Antibodies and Antibody-Based Screening Methods

The invention provides isolated or recombinant antibodies thatspecifically bind to an amylase of the invention. These antibodies canbe used to isolate, identify or quantify the amylases of the inventionor related polypeptides. These antibodies can be used to isolate otherpolypeptides within the scope the invention or other related amylases.The antibodies can be designed to bind to an active site of an amylase.Thus, the invention provides methods of inhibiting amylases using theantibodies of the invention.

The antibodies can be used in immunoprecipitation, staining,immunoaffinity columns, and the like. If desired, nucleic acid sequencesencoding for specific antigens can be generated by immunization followedby isolation of polypeptide or nucleic acid, amplification or cloningand immobilization of polypeptide onto an array of the invention.Alternatively, the methods of the invention can be used to modify thestructure of an antibody produced by a cell to be modified, e.g., anantibody's affinity can be increased or decreased. Furthermore, theability to make or modify antibodies can be a phenotype engineered intoa cell by the methods of the invention.

Methods of immunization, producing and isolating antibodies (polyclonaland monoclonal) are known to those of skill in the art and described inthe scientific and patent literature, see, e.g., Coligan, CURRENTPROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY (1991); Stites (eds.) BASICAND CLINICAL IMMUNOLOGY (7th ed.) Lange Medical Publications, Los Altos,Calif. (“Stites”); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES ANDPRACTICE (2d ed.) Academic Press, New York, N.Y. (1986); Kohler (1975)Nature 256:495; Harlow (1988) ANTIBODIES, A LABORATORY MANUAL, ColdSpring Harbor Publications, New York. Antibodies also can be generatedin vitro, e.g., using recombinant antibody binding site expressing phagedisplay libraries, in addition to the traditional in vivo methods usinganimals. See, e.g., Hoogenboom (1997) Trends Biotechnol. 15:62-70; Katz(1997) Annu. Rev. Biophys. Biomol. Struct. 26:27-45.

Polypeptides or peptides can be used to generate antibodies which bindspecifically to the polypeptides, e.g., the amylases, of the invention.The resulting antibodies may be used in immunoaffinity chromatographyprocedures to isolate or purify the polypeptide or to determine whetherthe polypeptide is present in a biological sample. In such procedures, aprotein preparation, such as an extract, or a biological sample iscontacted with an antibody capable of specifically binding to one of thepolypeptides of the invention.

In immunoaffinity procedures, the antibody is attached to a solidsupport, such as a bead or other column matrix. The protein preparationis placed in contact with the antibody under conditions in which theantibody specifically binds to one of the polypeptides of the invention.After a wash to remove non-specifically bound proteins, the specificallybound polypeptides are eluted.

The ability of proteins in a biological sample to bind to the antibodymay be determined using any of a variety of procedures familiar to thoseskilled in the art. For example, binding may be determined by labelingthe antibody with a detectable label such as a fluorescent agent, anenzymatic label, or a radioisotope. Alternatively, binding of theantibody to the sample may be detected using a secondary antibody havingsuch a detectable label thereon. Particular assays include ELISA assays,sandwich assays, radioimmunoassays, and Western Blots.

Polyclonal antibodies generated against the polypeptides of theinvention can be obtained by direct injection of the polypeptides intoan animal or by administering the polypeptides to a non-human animal.The antibody so obtained will then bind the polypeptide itself. In thismanner, even a sequence encoding only a fragment of the polypeptide canbe used to generate antibodies which may bind to the whole nativepolypeptide. Such antibodies can then be used to isolate the polypeptidefrom cells expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique, the trioma technique, thehuman B-cell hybridoma technique, and the EBV-hybridoma technique (see,e.g., Cole (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (see,e.g., U.S. Pat. No. 4,946,778) can be adapted to produce single chainantibodies to the polypeptides of the invention. Alternatively,transgenic mice may be used to express humanized antibodies to thesepolypeptides or fragments thereof.

Antibodies generated against the polypeptides of the invention may beused in screening for similar polypeptides (e.g., amylases) from otherorganisms and samples. In such techniques, polypeptides from theorganism are contacted with the antibody and those polypeptides whichspecifically bind the antibody are detected. Any of the proceduresdescribed above may be used to detect antibody binding.

Kits

The invention provides kits comprising the compositions, e.g., nucleicacids, expression cassettes, vectors, cells, transgenic seeds or plantsor plant parts, polypeptides (e.g., amylases) and/or antibodies of theinvention. The kits also can contain instructional material teaching themethodologies and industrial uses of the invention, as described herein.

Measuring Metabolic Parameters

The methods of the invention provide whole cell evolution, or whole cellengineering, of a cell to develop a new cell strain having a newphenotype, e.g., a new or modified amylase activity, by modifying thegenetic composition of the cell. The genetic composition can be modifiedby addition to the cell of a nucleic acid of the invention. To detectthe new phenotype, at least one metabolic parameter of a modified cellis monitored in the cell in a “real time” or “on-line” time frame. Inone aspect, a plurality of cells, such as a cell culture, is monitoredin “real time” or “on-line.” In one aspect, a plurality of metabolicparameters is monitored in “real time” or “on-line.” Metabolicparameters can be monitored using the amylases of the invention.

Metabolic flux analysis (MFA) is based on a known biochemistryframework. A linearly independent metabolic matrix is constructed basedon the law of mass conservation and on the pseudo-steady statehypothesis (PSSH) on the intracellular metabolites. In practicing themethods of the invention, metabolic networks are established, includingthe:

identity of all pathway substrates, products and intermediarymetabolites

identity of all the chemical reactions interconverting the pathwaymetabolites, the stoichiometry of the pathway reactions,

identity of all the enzymes catalyzing the reactions, the enzymereaction kinetics,

the regulatory interactions between pathway components, e.g. allostericinteractions, enzyme-enzyme interactions etc,

intracellular compartmentalization of enzymes or any othersupramolecular organization of the enzymes, and,

the presence of any concentration gradients of metabolites, enzymes oreffector molecules or diffusion barriers to their movement.

Once the metabolic network for a given strain is built, mathematicpresentation by matrix notion can be introduced to estimate theintracellular metabolic fluxes if the on-line metabolome data isavailable. Metabolic phenotype relies on the changes of the wholemetabolic network within a cell. Metabolic phenotype relies on thechange of pathway utilization with respect to environmental conditions,genetic regulation, developmental state and the genotype, etc. In oneaspect of the methods of the invention, after the on-line MFAcalculation, the dynamic behavior of the cells, their phenotype andother properties are analyzed by investigating the pathway utilization.For example, if the glucose supply is increased and the oxygen decreasedduring the yeast fermentation, the utilization of respiratory pathwayswill be reduced and/or stopped, and the utilization of the fermentativepathways will dominate. Control of physiological state of cell cultureswill become possible after the pathway analysis. The methods of theinvention can help determine how to manipulate the fermentation bydetermining how to change the substrate supply, temperature, use ofinducers, etc. to control the physiological state of cells to move alongdesirable direction. In practicing the methods of the invention, the MFAresults can also be compared with transcriptome and proteome data todesign experiments and protocols for metabolic engineering or geneshuffling, etc.

In practicing the methods of the invention, any modified or newphenotype can be conferred and detected, including new or improvedcharacteristics in the cell. Any aspect of metabolism or growth can bemonitored.

Monitoring Expression of an mRNA Transcript

In one aspect of the invention, the engineered phenotype comprisesincreasing or decreasing the expression of an mRNA transcript (e.g., anamylase message) or generating new (e.g., amylase) transcripts in acell. This increased or decreased expression can be traced by testingfor the presence of an amylase of the invention or by amylase activityassays. mRNA transcripts, or messages, also can be detected andquantified by any method known in the art, including, e.g., Northernblots, quantitative amplification reactions, hybridization to arrays,and the like. Quantitative amplification reactions include, e.g.,quantitative PCR, including, e.g., quantitative reverse transcriptionpolymerase chain reaction, or RT-PCR; quantitative real time RT-PCR, or“real-time kinetic RT-PCR” (see, e.g., Kreuzer (2001) Br. J. Haematol.114:313-318; Xia (2001) Transplantation 72:907-914).

In one aspect of the invention, the engineered phenotype is generated byknocking out expression of a homologous gene. The gene's coding sequenceor one or more transcriptional control elements can be knocked out,e.g., promoters or enhancers. Thus, the expression of a transcript canbe completely ablated or only decreased.

In one aspect of the invention, the engineered phenotype comprisesincreasing the expression of a homologous gene. This can be effected byknocking out of a negative control element, including a transcriptionalregulatory element acting in cis- or trans-, or, mutagenizing a positivecontrol element. One or more, or, all the transcripts of a cell can bemeasured by hybridization of a sample comprising transcripts of thecell, or, nucleic acids representative of or complementary totranscripts of a cell, by hybridization to immobilized nucleic acids onan array.

Monitoring Expression of a Polypeptides, Peptides and Amino Acids

In one aspect of the invention, the engineered phenotype comprisesincreasing or decreasing the expression of a polypeptide (e.g., anamylase) or generating new polypeptides in a cell. This increased ordecreased expression can be traced by determining the amount of amylasepresent or by amylase activity assays. Polypeptides, peptides and aminoacids also can be detected and quantified by any method known in theart, including, e.g., nuclear magnetic resonance (NMR),spectrophotometry, radiography (protein radiolabeling), electrophoresis,capillary electrophoresis, high performance liquid chromatography(HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography,various immunological methods, e.g. immunoprecipitation,immunodiffusion, immuno-electrophoresis, radioimmunoassays (RIAs),enzyme-linked immunosorbent assays (ELISAs), immuno-fluorescent assays,gel electrophoresis (e.g., SDS-PAGE), staining with antibodies,fluorescent activated cell sorter (FACS), pyrolysis mass spectrometry,Fourier-Transform Infrared Spectrometry, Raman spectrometry, GC-MS, andLC-Electrospray and cap-LC-tandem-electrospray mass spectrometries, andthe like. Novel bioactivities can also be screened using methods, orvariations thereof, described in U.S. Pat. No. 6,057,103. Furthermore,as discussed below in detail, one or more, or, all the polypeptides of acell can be measured using a protein array.

INDUSTRIAL APPLICATIONS

Detergent Compositions

The invention provides detergent compositions comprising one or morepolypeptides of the invention, and methods of making and using thesecompositions. The invention incorporates all methods of making and usingdetergent compositions, see, e.g., U.S. Pat. Nos. 6,413,928; 6,399,561;6,365,561; 6,380,147. The detergent compositions can be a one and twopart aqueous composition, a non-aqueous liquid composition, a castsolid, a granular form, a particulate form, a compressed tablet, a geland/or a paste and a slurry form. The invention also provides methodscapable of a rapid removal of gross food soils, films of food residueand other minor food compositions using these detergent compositions.Amylases of the invention can facilitate the removal of starchy stainsby means of catalytic hydrolysis of the starch polysaccharide. Amylasesof the invention can be used in dishwashing detergents in textilelaundering detergents.

The actual active enzyme content depends upon the method of manufactureof a detergent composition and is not critical, assuming the detergentsolution has the desired enzymatic activity. In one aspect, the amountof amylase present in the final solution ranges from about 0.001 mg to0.5 mg per gram of the detergent composition. The particular enzymechosen for use in the process and products of this invention dependsupon the conditions of final utility, including the physical productform, use pH, use temperature, and soil types to be degraded or altered.The enzyme can be chosen to provide optimum activity and stability forany given set of utility conditions. In one aspect, the polypeptides ofthe present invention are active in the pH ranges of from about 4 toabout 12 and in the temperature range of from about 20° C. to about 95°C. The detergents of the invention can comprise cationic, semi-polarnonionic or zwitterionic surfactants; or, mixtures thereof.

Amylases of the present invention can be formulated into powdered andliquid detergents having pH between 4.0 and 12.0 at levels of about 0.01to about 5% (preferably 0.1% to 0.5%) by weight. These detergentcompositions can also include other enzymes such as known proteases,cellulases, lipases or endoglycosidases, as well as builders andstabilizers. The addition of amylases of the invention to conventionalcleaning compositions does not create any special use limitation. Inother words, any temperature and pH suitable for the detergent is alsosuitable for the present compositions as long as the pH is within theabove range, and the temperature is below the described enzyme'sdenaturing temperature. In addition, the polypeptides of the inventioncan be used in a cleaning composition without detergents, again eitheralone or in combination with builders and stabilizers.

The present invention provides cleaning compositions including detergentcompositions for cleaning hard surfaces, detergent compositions forcleaning fabrics, dishwashing compositions, oral cleaning compositions,denture cleaning compositions, and contact lens cleaning solutions.

In one aspect, the invention provides a method for washing an objectcomprising contacting the object with a polypeptide of the inventionunder conditions sufficient for washing. A polypeptide of the inventionmay be included as a detergent additive. The detergent composition ofthe invention may, for example, be formulated as a hand or machinelaundry detergent composition comprising a polypeptide of the invention.A laundry additive suitable for pre-treatment of stained fabrics cancomprise a polypeptide of the invention. A fabric softener compositioncan comprise a polypeptide of the invention. Alternatively, apolypeptide of the invention can be formulated as a detergentcomposition for use in general household hard surface cleaningoperations. In alternative aspects, detergent additives and detergentcompositions of the invention may comprise one or more other enzymessuch as a protease, a lipase, a cutinase, another amylase, acarbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, agalactanase, a xylanase, an oxidase, e.g., a lactase, and/or aperoxidase. The properties of the enzyme(s) of the invention are chosento be compatible with the selected detergent (i.e. pH-optimum,compatibility with other enzymatic and non-enzymatic ingredients, etc.)and the enzyme(s) is present in effective amounts. In one aspect,amylase enzymes of the invention are used to remove malodorous materialsfrom fabrics. Various detergent compositions and methods for making themthat can be used in practicing the invention are described in, e.g.,U.S. Pat. Nos. 6,333,301; 6,329,333; 6,326,341; 6,297,038; 6,309,871;6,204,232; 6,197,070; 5,856,164.

Treating Fabrics

The invention provides methods of treating fabrics using one or morepolypeptides of the invention. The polypeptides of the invention can beused in any fabric-treating method, which are well known in the art,see, e.g., U.S. Pat. No. 6,077,316. For example, in one aspect, the feeland appearance of a fabric is improved by a method comprising contactingthe fabric with an amylase of the invention in a solution. In oneaspect, the fabric is treated with the solution under pressure.

In one aspect, the enzymes of the invention are applied during or afterthe weaving of textiles, or during the desizing stage, or one or moreadditional fabric processing steps. During the weaving of textiles, thethreads are exposed to considerable mechanical strain. Prior to weavingon mechanical looms, warp yarns are often coated with sizing starch orstarch derivatives in order to increase their tensile strength and toprevent breaking. The enzymes of the invention can be applied to removethese sizing starch or starch derivatives. After the textiles have beenwoven, a fabric can proceed to a desizing stage. This can be followed byone or more additional fabric processing steps. Desizing is the act ofremoving size from textiles. After weaving, the size coating must beremoved before further processing the fabric in order to ensure ahomogeneous and wash-proof result. The invention provides a method ofdesizing comprising enzymatic hydrolysis of the size by the action of anenzyme of the invention.

The enzymes of the invention can be used to desize fabrics, includingcotton-containing fabrics, as detergent additives, e.g., in aqueouscompositions. The invention provides methods for producing a stonewashedlook on indigo-dyed denim fabric and garments. For the manufacture ofclothes, the fabric can be cut and sewn into clothes or garments, thatis afterwards finished. In particular, for the manufacture of denimjeans, different enzymatic finishing methods have been developed. Thefinishing of denim garment normally is initiated with an enzymaticdesizing step, during which garments are subjected to the action ofamylolytic enzymes in order to provide softness to the fabric and makethe cotton more accessible to the subsequent enzymatic finishing steps.The invention provides methods of finishing denim garments (e.g., a“bio-stoning process”), enzymatic desizing and providing softness tofabrics using the amylases of the invention. The invention providesmethods for quickly softening denim garments in a desizing and/orfinishing process.

Foods and Food Processing

The enzymes of the invention have numerous applications in foodprocessing industry. The amylases of the invention are used in starch tofructose processing. Starch to fructose processing can consist of foursteps: liquefaction of granular starch, saccharification of theliquefied starch into dextrose, purification, and isomerization tofructose.

The invention provides methods of starch liquefaction using the enzymesof the invention. Concentrated suspensions of starch polymer granulesare converted into a solution of soluble shorter chain length dextrinsof low viscosity. This step is useful for convenient handling withstandard equipment and for efficient conversion to glucose or 10³ othersugars. In one aspect, the granular starch is liquefied by gelatinizingthe granules by raising the temperature of the granular starch to overabout 72° C. The heating process instantaneously disrupts the insolublestarch granules to produce a water soluble starch solution. Thesolubilized starch solution can then be liquefied by an amylase of theinvention. Thus, the invention provides enzymatic starch liquefactionprocesses using an amylase of the invention.

An exemplary enzymatic liquefaction process involves adjusting the pH ofa granular starch slurry to between 6.0 and 6.5 and the addition ofcalcium hydroxide, sodium hydroxide or sodium carbonate. In one aspect,calcium hydroxide is added. This provides calcium ions to stabilize theglucoamylase of the invention against inactivation. In one aspect, uponaddition of amylase, the suspension is pumped through a steam jet toinstantaneously raise the temperature to between 80°-415° C. In oneaspect, the starch is immediately gelatinized and, due to the presenceof amylase, depolymerized through random hydrolysis of α-1,4-glycosidicbonds by amylase to a fluid mass. The fluid mass can be easily pumped.

The invention provides various enzymatic starch liquefaction processesusing an amylase of the invention. In one aspect of the liquefactionprocess of the invention, an amylase is added to the starch suspensionand the suspension is held at a temperature of between about 80°-100° C.to partially hydrolyze the starch granules. In one aspect, the partiallyhydrolyzed starch suspension is pumped through a jet at temperatures inexcess of about 105° C. to thoroughly gelatinize any remaining granularstructure. In one aspect, after cooling the gelatinized starch, a secondaddition of amylase is made to further hydrolyze the starch.

The invention provides enzymatic dry milling processes using an amylaseof the invention. In dry milling, whole grain is ground and combinedwith water. The germ is optionally removed by flotation separation orequivalent techniques. The resulting mixture, which contains starch,fiber, protein and other components of the grain, is liquefied usingamylase. In one aspect, enzymatic liquefaction is done at lowertemperatures than the starch liquification processes discussed above. Inone aspect, after gelatinization the starch solution is held at anelevated temperature in the presence of amylase until a DE of 10-20 isachieved. In one aspect, this is a period of about 1-3 hours. Dextroseequivalent (DE) is the industry standard for measuring the concentrationof total reducing sugars, calculated as D-glucose on a dry weight basis.Unhydrolyzed granular starch has a DE of virtually zero, whereas the DEof D-glucose is defined as 100.

The invention provides wet milling processes, e.g., corn wet milling,using an amylase of the invention. Corn wet milling is a process whichproduces corn oil, gluten meal, gluten feed and starch. Thus, theinvention provides methods of making corn oil, gluten meal, gluten feedand starch using an enzyme of the invention. In one aspect, analkaline-amylase of the invention is used in the liquefaction of starch.In one aspect, glucoamylase is used in saccharification to produceglucose.

In one aspect, corn (a kernel that consists of a outer seed coat(fiber), starch, a combination of starch and glucose and the innergerm), is subjected to a four step process, which results in theproduction of starch. In one aspect, the corn is steeped, de-germed,de-fibered, and the gluten is separated. In a steeping process thesolubles are taken out. The product remaining after removal of thesolubles is de-germed, resulting in production of corn oil andproduction of an oil cake, which is added to the solubles from thesteeping step. The remaining product is de-fibered and the fiber solidsare added to the oil cake/solubles mixture. This mixture of fibersolids, oil cake and solubles forms a gluten feed. After de-fibering,the remaining product is subjected to gluten separation. This separationresults in a gluten meal and starch. The starch is then subjected toliquefaction and saccharification using polypeptides of the invention toproduce glucose.

The invention provides anti-staling processes (e.g., of baked productssuch as bread) using an amylase of the invention. The invention providesmethods to slow the increase of the firmness of the crumb (of the bakedproduct) and a decrease of the elasticity of the crumb using an amylaseof the invention. Staling of baked products (such as bread) is moreserious as time passes between the moment of preparation of the breadproduct and the moment of consumption. The term staling is used todescribe changes undesirable to the consumer in the properties of thebread product after leaving the oven, such as an increase of thefirmness of the crumb, a decrease of the elasticity of the crumb, andchanges in the crust, which becomes tough and leathery. The firmness ofthe bread crumb increases further during storage up to a level, which isconsidered as negative. Amylases of the invention are used to retardstaling of the bread as described e.g., in U.S. Pat. Nos. 6,197,352;2,615,810 and 3,026,205; Silberstein (1964) Baker's Digest 38:66-72.

In one aspect, an enzyme of the invention is used to retard the stalingof baked products while not hydrolyzing starch into the brancheddextrins. Branched dextrins are formed by cleaving off the branchedchains of the dextrins generated by α-amylase hydrolysis which cannot bedegraded further by the α-amylase. This can produce a gummy crumb in theresulting bread. Accordingly, the invention provides a process forretarding the staling of baked products (e.g., leavened baked products)comprising adding an enzyme of the invention comprising exoamylaseactivity to a flour or a dough used for producing a baked product.Exoamylases of the invention can have glucoamylase, β-amylase (whichreleases maltose in the beta-configuration) and/or maltogenic amylaseactivity.

The invention also provides a process for preparing a dough or a bakedproduct prepared from the dough which comprises adding an amylase of theinvention to the dough in an amount which is effective to retard thestaling of the bread. The invention also provides a dough comprisingsaid amylase and a premix comprising flour together with said amylase.Finally, the invention provides an enzymatic baking additive, whichcontains said amylase.

The invention also provides a high yield process for producing highquality corn fiber gum by treatment of corn fiber with an enzyme of theinvention followed by hydrogen peroxide treatment to obtain an extractof milled corn fiber. See, e.g., U.S. Pat. No. 6,147,206.

Animal Feeds and Additives

The invention provides methods for treating animal feeds and additivesusing amylase enzymes of the invention. The invention provides animalfeeds and additives comprising amylases of the invention. In one aspect,treating animal feeds and additives using amylase enzymes of theinvention can help in the availability of starch in the animal feed oradditive. This can result in release of readily digestible and easilyabsorbed sugars.

Use of an amylase of the invention can increase the digestive capacityof animals and birds. Use of an amylase of the invention can ensureavailability of an adequate nutrient supply for better growth andperformance. In one aspect, the enzymes of the invention can be added asfeed additives for animals. In another aspect, the animal feed can betreated with amylases prior to animal consumption. In another aspect,the amylases may be supplied by expressing the enzymes directly intransgenic feed crops (as, e.g., transgenic plants, seeds and the like),such as corn. As discussed above, the invention provides transgenicplants, plant parts and plant cells comprising a nucleic acid sequenceencoding a polypeptide of the invention. In one aspect, the nucleic acidis expressed such that the amylase is produced in recoverablequantities. The amylase can be recovered from any plant or plant part.Alternatively, the plant or plant part containing the recombinantpolypeptide can be used as such for improving the quality of a food orfeed, e.g., improving nutritional value, palatability, and rheologicalproperties, or to destroy an antinutritive factor.

Paper or Pulp Treatment

The enzymes of the invention can be in paper or pulp treatment or paperdeinking. For example, in one aspect, the invention provides a papertreatment process using amylases of the invention. In one aspect, theenzymes of the invention can be used to modify starch in the paperthereby converting it into a liquefied form. In another aspect, papercomponents of recycled photocopied paper during chemical and enzymaticdeinking processes. In one aspect, amylases of the invention can be usedin combination with cellulases. The paper can be treated by thefollowing three processes: 1) disintegration in the presence of anenzyme of the invention, 2) disintegration with a deinking chemical andan enzyme of the invention, and/or 3) disintegration after soaking withan enzyme of the invention. The recycled paper treated with amylase canhave a higher brightness due to removal of toner particles as comparedto the paper treated with just cellulase. While the invention is notlimited by any particular mechanism, the effect of an amylase of theinvention may be due to its behavior as surface-active agents in pulpsuspension.

The invention provides methods of treating paper and paper pulp usingone or more polypeptides of the invention. The polypeptides of theinvention can be used in any paper- or pulp-treating method, which arewell known in the art, see, e.g., U.S. Pat. Nos. 6,241,849; 6,066,233;5,582,681. For example, in one aspect, the invention provides a methodfor deinking and decolorizing a printed paper containing a dye,comprising pulping a printed paper to obtain a pulp slurry, anddislodging an ink from the pulp slurry in the presence of an enzyme ofthe invention (other enzymes can also be added). In another aspect, theinvention provides a method for enhancing the freeness of pulp, e.g.,pulp made from secondary fiber, by adding an enzymatic mixturecomprising an enzyme of the invention (can also include other enzymes,e.g., pectinase enzymes) to the pulp and treating under conditions tocause a reaction to produce an enzymatically treated pulp. The freenessof the enzymatically treated pulp is increased from the initial freenessof the secondary fiber pulp without a loss in brightness.

Repulping: Treatment of Lignocellulosic Materials

The invention also provides a method for the treatment oflignocellulosic fibers, wherein the fibers are treated with apolypeptide of the invention, in an amount which is efficient forimproving the fiber properties. The amylases of the invention may alsobe used in the production of lignocellulosic materials such as pulp,paper and cardboard, from starch reinforced waste paper and cardboard,especially where repulping occurs at pH above 7 and where amylases canfacilitate the disintegration of the waste material through degradationof the reinforcing starch. The amylases of the invention can be usefulin a process for producing a papermaking pulp from starch-coated printedpaper. The process may be performed as described in, e.g., WO 95/14807.

An exemplary process comprises disintegrating the paper to produce apulp, treating with a starch-degrading enzyme before, during or afterthe disintegrating, and separating ink particles from the pulp afterdisintegrating and enzyme treatment. See also U.S. Pat. No. 6,309,871and other US patents cited herein. Thus, the invention includes a methodfor enzymatic deinking of recycled paper pulp, wherein the polypeptideis applied in an amount which is efficient for effective de-inking ofthe fiber surface.

Waste Treatment

The enzymes of the invention can be used in a variety of otherindustrial applications, e.g., in waste treatment. For example, in oneaspect, the invention provides a solid waste digestion process usingenzymes of the invention. The methods can comprise reducing the mass andvolume of substantially untreated solid waste. Solid waste can betreated with an enzymatic digestive process in the presence of anenzymatic solution (including an enzyme of the invention) at acontrolled temperature. This results in a reaction without appreciablebacterial fermentation from added microorganisms. The solid waste isconverted into a liquefied waste and any residual solid waste. Theresulting liquefied waste can be separated from said any residualsolidified waste. See e.g., U.S. Pat. No. 5,709,796.

Oral Care Products

The invention provides oral care product comprising an amylase of theinvention. Exemplary oral care products include toothpastes, dentalcreams, gels or tooth powders, odontics, mouth washes, pre- or postbrushing rinse formulations, chewing gums, lozenges, or candy. See,e.g., U.S. Pat. No. 6,264,925.

Brewing and Fermenting

The invention provides methods of brewing (e.g., fermenting) beercomprising an amylase of the invention. In one exemplary process,starch-containing raw materials are disintegrated and processed to forma malt. An amylase of the invention is used at any point in thefermentation process. For example, amylases of the invention can be usedin the processing of barley malt. The major raw material of beer brewingis barley malt. This can be a three stage process. First, the barleygrain can be steeped to increase water content, e.g., to around about40%. Second, the grain can be germinated by incubation at 15-25° C. for3 to 6 days when enzyme synthesis is stimulated under the control ofgibberellins. During this time amylase levels rise significantly. In oneaspect, amylases of the invention are added at this (or any other) stageof the process. The action of the amylase results in an increase infermentable reducing sugars. This can be expressed as the diastaticpower, DP, which can rise from around 80 to 190 in 5 days at 12° C.

Amylases of the invention can be used in any beer producing process, asdescribed, e.g., in U.S. Pat. Nos. 5,762,991; 5,536,650; 5,405,624;5,021,246; 4,788,066.

OTHER INDUSTRIAL APPLICATIONS

The invention also includes a method of increasing the flow ofproduction fluids from a subterranean formation by removing a viscous,starch-containing, damaging fluid formed during production operationsand found within the subterranean formation which surrounds a completedwell bore comprising allowing production fluids to flow from the wellbore; reducing the flow of production fluids from the formation belowexpected flow rates; formulating an enzyme treatment by blendingtogether an aqueous fluid and a polypeptide of the invention; pumpingthe enzyme treatment to a desired location within the well bore;allowing the enzyme treatment to degrade the viscous, starch-containing,damaging fluid, whereby the fluid can be removed from the subterraneanformation to the well surface; and wherein the enzyme treatment iseffective to attack the alpha glucosidic linkages in thestarch-containing fluid.

In summary, the invention provides enzymes and processes for hydrolyzingliquid (liquefied) and granular starch. Such starch can be derived fromany source, e.g., corn, wheat, milo, sorghum, rye or bulgher. Theinvention applies to any grain starch source which is useful inliquefaction, e.g., any other grain or vegetable source known to producestarch suitable for liquefaction. The methods of the invention compriseliquefying starch from any natural material, such as rice, germinatedrice, corn, barley, milo, wheat, legumes and sweet potato. Theliquefying process can substantially hydrolyze the starch to produce asyrup. The temperature range of the liquefaction can be any liquefactiontemperature which is known to be effective in liquefying starch. Forexample, the temperature of the starch can be between about 80° C. toabout 115° C., between about 100° C. to about 110° C., and from about105° C. to about 108° C.

In other aspects, amylases of the invention can be used in biodefense(e.g., destruction of spores or bacteria). Use of amylases in biodefenseapplications offer a significant benefit, in that they can be veryrapidly developed against any currently unknown biological warfareagents of the future. In addition, amylases of the invention can be usedfor decontamination of affected environments. Additionally, amylases ofthe invention can be used in biofilm degradation, in biomass conversionto ethanol, and/or in the personal care and cosmetic industry.

EXAMPLES Example 1 Exemplary Protocol for Liquefying Starch andMeasuring Results

The following example described and exemplary protocol for liquefyingstarch using selected amylases of the invention.

Amylases having a sequence as set forth in SEQ ID NO:10 and SEQ ID NO:4demonstrated activity on liquefied starch at pH 4.5 or 6.5 using thereaction conditions show below.

Reaction Conditions: 100 mM PO₄ pH 6.5, 1% (w/w) liquefied starch DE 12at 55° C. Both TLC and HPLC assays were done to verify activity. Thedata from both assays showed that the clones were active.

pH profiles for the amylases having a sequence as set forth in SEQ IDNO:4 and SEQ ID NO:10 were run using phosphate buffer pHed from 3.0-6.5,at 55° C. From the amount of observable hydrolysis, it could be visuallysaid that the clones were more active at certain pH values than at othervalues at the above indicated reaction conditions:

SEQ ID NO:4—active from pH 5.0-6.5

SEQ ID NO:10—active from pH 4.5-6.5

An exemplary protocol for the saccharification of liquefied starch at pH6.5:

-   -   Adjust the pH of the liquefied starch to the pH at which the        saccharification(s) will be performed. Liquefy starch in 100 mM        sodium acetate buffer, pH 4.5 with 100 mM sodium phosphate salts        added so that before saccharification, the pH could be adjusted        to pH 6.5.    -   Weigh 5 gram samples of liquefied starch into tared bottles.    -   Use 0.04% (w/w) Optidex L-400 or approximately 400 mL of 1-10        diluted stock Optidex L-400 per 100 grams of liquefied starch.    -   Calculate the milligrams of Optidex L-400 contained in the 400        mL of 1-10 diluted stock Optidex L-400. Next, calculate the        volume of lysates needed to give the same concentration of        enzyme as the Optidex L-400.    -   Add enzymes to liquefied starch samples and incubate at desired        temperature (50 C.°). After 18 hours determine DE and prepare a        sample for HPLC analysis.

An exemplary DE Determination:

Exemplary Neocuproine Assay:

A 100 ml sample was added to 2.0 ml of neocuproine solution A (40 g/Lsodium carbonate, 16 g/L glycine, 0.45 g/L copper sulfate). To this wasadded 2.0 ml of neocuproine solution B (1.2 g/L neocuproinehydrochloride-Sigma N-1626). The tubes were mixed and heated in aboiling water bath for 12 minutes; cooled, diluted to 10 ml volume withDI water and the OD read at 450 nm on the spectrophotometer. The glucoseequivalent in the sample was extrapolated from the response of a 0.2mg/ml glucose standard run simultaneously.

Exemplary HPLC Analysis:

Saccharification carbohydrate profiles are measured by HPLC (Bio-RadAminex HPX-87A column in silver form, 80° C.) using refractive indexdetection. Mobile phase is filtered Millipore water used at a flow rateof 0.7 ml/min. Saccharification samples are diluted 1-10 with acidifiedDI water (5 drops of 6 M HCl into 200 mL DI water) then filtered througha 0.45 mm syringe filter. Injection volume is 20 uL.

Exemplary TLC:

Reaction products were w/d at hourly timepoints and spotted and dried ona TLC plate. The Plate was then developed in 10:90 water:isopropanol andvisualized with either a vanillin stain or CAM stain and then heated toshow reducible sugars. The liquefied starch was partially hydrolyzed toglucose in cases where activity was observed.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. An isolated, synthetic or recombinant polypeptide comprising (a) anamino acid sequence having at least 95% sequence identity to SEQ ID NO:8having glucomylase activity, or (b) said polypeptide encoded by anucleic acid having at least 95% sequence identity to SEQ ID NO:7.
 2. Afood supplement for an animal comprising the polypeptide as set forth inclaim
 1. 3. An edible enzyme delivery matrix comprising the polypeptideas set forth in claim
 1. 4. A method for hydrolyzing a starch or starchhydrolysate, comprising: contacting the polypeptide as set forth inclaim 1 with a composition comprising the starch under conditionswherein the polypeptide hydrolyzes the starch or starch hydrolysate. 5.A method for liquefying or removing a starch from a composition,comprising: contacting the polypeptide as set forth in claim 1 with thecomposition comprising the starch under conditions wherein thepolypeptide liquefies or removes the starch.
 6. A composition comprisingthe polypeptide as set forth in claim
 1. 7. A method for hydrolyzing astarch in a feed or food prior to consumption by an animal, comprising:(a) obtaining the feed or food comprising the starch; and (b) adding thepolypeptide as set forth in claim 1 to the feed or food in an amount andfor a time sufficient for the polypeptide to hydrolyze the starch in thefeed or food prior to consumption by the animal.
 8. The method as setforth in claim 7, wherein the feed or food comprises rice, corn, barley,wheat, legumes, or potato.
 9. A method for desizing a fabric,comprising: contacting the polypeptide as set forth in claim 1 with thefabric under conditions in which hydrolysis of starch in the fabric bythe polypeptide results in desizing the fabric.
 10. A method fordeinking a paper or fibers, comprising: contacting the polypeptide asset forth in claim 1 with the paper or fibers under conditions in whichhydrolysis of starch in the paper or fibers by the polypeptide resultsin deinking the fabric.
 11. A method for improving the properties oflignocellulosic fibers, comprising: contacting the polypeptide as setforth in claim 1 with the lignocellulosic fibers under conditions inwhich hydrolysis of starch in the lignocellulosic fibers by thepolypeptide improve the properties of the lignocellulosic fibers.
 12. Amethod for producing a high-maltose or high-glucose syrup, comprising:contacting the polypeptide as set forth in claim 1 and a compositioncomprising a starch or a starch hydrolysate under conditions in whichhydrolysis of starch or starch hydrolysate in the composition by thepolypeptide produces a high-maltose or a high-glucose syrup.
 13. Themethod as set forth in claim 12, wherein the starch is from rice, corn,barley, wheat, legumes, potato, or sweet potato.
 14. A method forimproving the flow of a starch-containing production fluid, comprising:contacting the polypeptide as set forth in claim 1 and thestarch-containing production fluid under conditions in which hydrolysisof starch in the production fluid by the polypeptide improves the flowof the fluid by decreasing the density of the fluid.
 15. The method asset forth in claim 14, wherein the production fluid is from asubterranean formation.
 16. An anti-staling composition comprisingpolypeptide as set forth in claim
 1. 17. A method for preventing stalingof a baked product, comprising: combining the polypeptide as set forthin claim 1 with a baked product comprising starch under conditions inwhich hydrolysis of starch in the baked product prevents staling of thebaked product.
 18. The method as set forth in claim 17, wherein thebaked product is bread.
 19. A method for alcohol production, comprising:combining the polypeptide as set forth in claim 1 with a compositioncomprising a starch or a starch hydrolysate under conditions in whichthe polypeptide hydrolyzes the starch or a starch hydrolysate; and usingthe resulting hydrolysate for alcohol production.
 20. A method forbrewing, comprising: combining the polypeptide as set forth in claim 1with a composition comprising a starch or a starch hydrolysate underconditions in which the polypeptide hydrolyzes the starch or a starchhydrolysate; and using the resulting hydrolysate for brewing.
 21. Themethod as set forth in claim 20, wherein the composition comprising thestarch is in a wort used for brewing beer.
 22. A method forsaccharification, comprising: combining a starch hydrolysate with thepolypeptide as set forth in claim 1 under conditions in which thepolypeptide saccharifies the starch hydrolysate.